Protein Crystallization Droplet Actuator, System and Method

ABSTRACT

Protein crystallization droplet actuators, systems and methods are provided. According to one embodiment, a droplet actuator for providing an array of crystallization conditions is provided and includes: (a) two or more processing reservoirs; (b) two or more dispensing reservoirs collectively comprising two or more crystallization reagents; and (c) a port for introducing a sample for crystallization analysis. Systems including the droplet actuator, methods of providing an array of crystallization conditions, and methods of identifying crystallization conditions are also provided.

2 RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2007/09379, entitled “Droplet-Based Multiwell Operations,”filed Apr. 18, 2007, pending, which claims the benefit of, is relatedto, and incorporates by reference related provisional U.S. PatentApplication Nos. 60/745,049, entitled “Apparatus and Methods forDroplet-Based Protein Crystallization,” filed on Apr. 18, 2006;60/745,054, entitled “Droplet-Based Multi-Well Plate,” filed on Apr. 18,2006; and 60/806,400, entitled “Droplet-Microactuator StampingPlatform,” filed on Jun. 30, 2006.

1 GRANT INFORMATION

The work leading to this invention was supported at least in part by NIHGrant Nos. 1 R43 GM072155-01 and 2 R44 GM072155-02. The United StatesGovernment may have certain rights in the invention described herein.

3 FIELD OF THE INVENTION

The present invention broadly relates to a protein crystallizationdroplet actuator, system and method. Embodiments of the presentinvention relate to a droplet actuator for providing an array ofcrystallization conditions, and associated systems and methods.

4 BACKGROUND OF THE INVENTION

4.1 Protein Crystallization Approaches

Protein expression and purification is an expensive process, and it issometimes difficult to express proteins in large quantities. A largeamount of expensive protein is required to establish conditions forcrystallization. Consequently, there is a need to reduce the amount ofprotein required for crystallization screening and to do it moreefficiently and at lower cost.

4.1.1 Protein Crystal Synthesis

Proteins play a key role in all biological processes. The specificbiological function of a protein is determined by the three-dimensional(3D) arrangement of the constituent amino acids. Understanding aprotein's 3D structure plays an important role in protein engineering,bioseparations, rational drug design, controlled drug delivery, and thedesign of novel enzyme substrates, activators, and inhibitors. Proteincrystallization is a multi-parametric process that involves the steps ofnucleation and growth, during which molecules are brought into athermodynamically unstable and a supersaturated state.

4.1.2 Miniaturization and Automation of Protein Crystallization Setup

Many proteins of interest are unfortunately available only in limitedsupply. Efforts are ongoing to reduce the consumption of proteins byminiaturizing the crystallization setup. Despite efforts to reduce theprotein volumes, these processes still consume significant amount ofprotein and are still labor-intensive.

Existing semi-automatic systems do not encompass ideal high-throughputconfigurations. They require user intervention for multiple trayprocessing and have other material processing issues. As most of thework performed with these systems is not on a large scale, automation ofstorage and handling of plates was not addressed in these systems. Eventhough these industrial systems are capable of setting up thousands ofcrystallization screens a day, they are prohibitively expensive foracademic research labs. There remains a need in the art for a systemthat provides the high-throughput automation functionality of anindustrial system at an affordable cost for small laboratories orindividual investigators.

4.1.3 Lab-on-a-Chip Technologies

Microfluidic systems can be broadly categorized into continuous-flow anddiscrete-flow based systems. As the name suggests, continuous-flowsystems rely on continuous flow of liquids in channels whereasdiscrete-flow systems utilize droplets of liquid within channels or inan architecture without channels. A common limitation that continuousflow systems face is that liquid transport is physically confined tofixed channels. The transport mechanisms used are usuallypressure-driven by external pumps or electrokinetically-driven byhigh-voltages. These approaches involve complex channeling and requirelarge supporting instruments in the form of external valves or powersupplies. These restrictions make it difficult to achieve high degreesof functional integration and control in conventional continuous-flowsystems.

4.2 Multi-Well Plates

Microfluidic technologies are attracting attention in pharmaceuticalresearch, as miniaturization of assay volume and improvement ofautomation, throughput and precision become more critical in drugdiscovery research. Examples of recent microfluidic technologies andproducts include the Topaz™ system for protein crystallization fromFluidigm Corporation (San Francisco, Calif.), the LabChip® system fromCaliper Life Sciences (Hopkinton, Mass.), and the LabCD™ system fromTecan Systems Inc. (San Jose, Calif.), both for ADME. These systemsperform certain assays using small volumes of liquid. However, none ofthem even remotely approaches the flexibility of conventional roboticsystems. This inadequacy results from inherent technical limitationsassociated with the way in which fluid handling is implemented in thesedevices.

Most existing technologies are based on a continuous-flow approach.Liquid is pumped (generally unidirectionally) through a network ofmicrochannels using external pumps, valves, high-voltage supplies orcentrifugal force. The primary disadvantage of all of thesecontinuous-flow microfluidic devices is their architectural andoperational rigidity. Most are optimized for a particular assay,providing little or no flexibility to make changes in reactionprotocols. The required continuity of fluid in these devices also makesindependent operation of different areas of the chip an inherentlydifficult proposition. Consequently, these technologies are non-modularand difficult to scale.

There is a need in the art for a microfluidic platform that avoids theuse of a continuous-flow approach. There is a need for a system thataffords flexibility and programmability that is comparable to roboticsystems. Further, there is a need for a system that is capable ofworking with droplets as small as a few nanoliters in volume and avoidsthe requirements for a network of microchannels, external pumps, valves,high-voltage supplies and/or centrifugal force. Further, there is a needfor a system that is scalable, permitting hundreds or even hundreds ofthousands of droplets of liquid to be processed in parallel. Finally,there is a need for a system that is both compact and inexpensive tomanufacture.

4.3 Protein Stamping Platforms

Mass spectroscopy (MS) is increasingly becoming the method of choice forprotein analysis in biological samples. Among the various MS methods,MALDI-TOF (Matrix Assisted Laser Desorption-Ionization Time of Flight)is the most commonly used due to its simplicity, high sensitivity andresolution. A typical MALDI-MS protocol for protein identificationinvolves sample preparation, stamping onto a MALDI target and analysison a MALDI-TOF mass spectrometer. Sample preparation steps (such asdigestion and concentration) are usually done in the well-plate formatand last for several hours at the least. The stamping is accomplishedusing complex robotic systems which are huge, expensive and immobile.Required sample volumes are also very high, which is a concern forproteins available in very small quantities. Existing microfluidicdevices are based on continuous flow in fixed microchannels, offeringvery little flexibility in terms of scalability and reconfigurability.As such, a need exists for a droplet microactuation stamping platformdesigned to solve the deficiencies found in the prior art.

5 BRIEF DESCRIPTION OF THE INVENTION

Embodiments of the present invention relate to a protein crystallizationdroplet actuator, system and method.

According to one embodiment, a droplet actuator for providing an arrayof crystallization conditions is provided and comprises: (a) two or moreprocessing reservoirs; (b) two or more dispensing reservoirscollectively comprising two or more crystallization reagents; and (c) aport for introducing a sample for crystallization analysis.

According to another embodiment, a system is provided and comprises thedroplet actuator as described above programmed to conduct dropletoperations sufficient to create an array of crystallization conditionsfor the sample on the droplet actuator.

According to yet another embodiment, a method of providing an array ofcrystallization conditions is provided and comprises: (a) providing adroplet actuator comprising: (i) two or more processing reservoirs; (ii)two or more dispensing reservoirs collectively comprising two or morecrystallization reagents; and (iii) a port for introducing a sample forcrystallization analysis; and (b) executing droplet operations on thedroplet actuator to combine crystallization reagents with sample in theprocessing reservoirs to create two or more crystallization conditionsin the processing reservoirs.

According to a further embodiment, a method of identifyingcrystallization conditions is provided and comprises: (a) providing anarray of crystallization conditions according to the method describedabove; (b) permitting the crystallization conditions to remain intactfor a predetermined period of time; and (c) identifying wells in whichcrystallization results.

6 DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Analyte,” means a target substance for detection which may be presentin a sample. Illustrative examples include antigenic substances,haptens, antibodies, proteins, peptides, amino acids, nucleotides,nucleic acids, drugs, ions, salts, small molecules, and cells.

“Bead,” with respect to beads on a droplet microactuator, means any beador particle capable of interacting with a droplet on or in proximitywith a droplet microactuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet microactuator;configured with respect to a droplet microactuator in a manner whichpermits a droplet on the droplet microactuator to be brought intocontact with the bead, on the droplet microactuator and/or off thedroplet microactuator. Beads may be manufactured using a wide variety ofmaterials, including for example, resins, and polymers. The beads may beany suitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onlyone component of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, “Multiplex flow assays preferably with magnetic particlesas solid phase,” published on Nov. 24, 2005, the entire disclosure ofwhich is incorporated herein by reference for its teaching concerningmagnetically responsive materials and beads.

“Communicate” (e.g., a first component “communicates with” or “is incommunication with” a second component) is used herein to indicate astructural, functional, mechanical, optical, electrical, or fluidicrelationship, or any combination thereof, between two or more componentsor elements. As such, the fact that one component is said to communicatewith a second component is not intended to exclude the possibility thatadditional components may be present between and/or operativelyassociated or engaged with, the first and second components.

“Chip” refers to any substrate including not only silicon orsemiconductors but glass, printed circuit boards, plastics or any othersubstrate on which the droplets are manipulated.

“Droplet” means a volume of liquid on a droplet microactuator which isat least partially bounded by filler fluid. For example, a droplet maybe completely surrounded by filler fluid or may be bounded by fillerfluid and one or more surfaces of the droplet microactuator. Dropletsmay take a wide variety of shapes; nonlimiting examples includegenerally disc shaped, slug shaped, truncated sphere, ellipsoid,spherical, partially compressed sphere, hemispherical, ovoid,cylindrical, and various shapes formed during droplet operations, suchas merging or splitting or formed as a result of contact of such shapeswith one or more surfaces of a droplet microactuator.

“Droplet operation” means any manipulation of a droplet on a dropletmicroactuator. A droplet operation may, for example, include: loading adroplet into the droplet microactuator; dispensing one or more dropletsfrom a source droplet; splitting, separating or dividing a droplet intotwo or more droplets; transporting a droplet from one location toanother in any direction; merging or combining two or more droplets intoa single droplet; diluting a droplet; mixing a droplet; agitating adroplet; deforming a droplet; retaining a droplet in position;incubating a droplet; heating a droplet; vaporizing a droplet; cooling adroplet; disposing of a droplet; transporting a droplet out of a dropletmicroactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading.

“Electronically coupled” is used herein to indicate an electrical ordata relationship between two or more components or elements. As such,the fact that a first component is said to be electronically coupled toa second component is not intended to exclude the possibility thatadditional components may be present between, and/or operativelyassociated or engaged with, the first and second components. Further,electrically coupled components may in some embodiments include wirelessintervening components.

“Input device” is used broadly to include all possible types of devicesand ways to input information into a computer system or onto a network.Examples include stylus-based devices, pen-based devices, keyboarddevices, keypad devices, touchpad devices, touch screen devices,joystick devices, trackball devices, mouse devices, bar-code readerdevices, magnetic strip reader devices, infrared devices, speechrecognition technologies.

“Output device” is used broadly to include all possible types of devicesand ways to output information or data from a computer system to a useror to another system. Examples include visual displays, LEDs, printers,speakers, modems and wireless transceivers.

“Protocol” means a series of steps that includes, but is not limited to,droplet operations on one or more droplet microactuators.

“Surface” with reference to immobilization of a molecule, such as anantibody or in analyte, on the surface, means any surface on which themolecule can be immobilized while retaining the capability to interactwith droplets on a droplet microactuator. For example, the surface maybe a surface on the droplet microactuator, such as a surface on the topplate or bottom plate of the droplet microactuator; a surface extendingfrom the top plate or bottom plate of the droplet microactuator; asurface on a physical object positioned on the droplet microactuator ina manner which permits it to interact with droplets on the dropletmicroactuator; and/or a bead positioned on the droplet microactuator,e.g., in a droplet and/or in a droplet microactuator but exterior to thedroplet.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet microactuatorfor convenience only, since the droplet microactuator is functionalregardless of its position in space.

When a given component such as a layer, region or substrate is referredto herein as being disposed or formed “on” another component, that givencomponent can be directly on the other component or, alternatively,intervening components (for example, one or more coatings, layers,interlayers, electrodes or contacts) can also be present. It will befurther understood that the terms “disposed on” and “formed on” are usedinterchangeably to describe how a given component is positioned orsituated in relation to another component. Hence, the terms “disposedon” and “formed on” are not intended to introduce any limitationsrelating to particular methods of material transport, deposition, orfabrication.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletmicroactuator, it should be understood that the droplet is arranged onthe droplet microactuator in a manner which facilitates using thedroplet microactuator to conduct droplet operations on the droplet, thedroplet is arranged on the droplet microactuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet microactuator.

7 BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a 96-well optimization chip inaccordance with an embodiment of the present invention;

FIG. 2 is graph of transport characteristics of different surfactantsolutions depicting the maximum droplet transfer frequency as a functionof voltage;

FIG. 3 is an illustration of lysozyme crystals formed on-chip at 20×magnification in accordance with an embodiment of the present invention;

FIG. 4 is an illustration of glucose isomerase crystals formed on-chipat 20× magnification in accordance with an embodiment of the presentinvention;

FIG. 5 is an illustration of proteinase K crystals formed on-chip at 40×magnification in accordance with an embodiment of the present invention;

FIGS. 6A-6D are illustrations of lysozyme crystals formed on-chip atvarious NaCl solutions in accordance with an embodiment of the presentinvention;

FIG. 7 is a plan view of wells and surrounding electrodes of amicrofluidic chip in accordance with an embodiment of the presentinvention;

FIG. 8 is a schematic plan view of a screening chip in accordance withan embodiment of the present invention;

FIG. 9 is a schematic plan view of an optimization chip in accordancewith an embodiment of the present invention;

FIG. 10 is a plan view of a microfluidic chip in accordance with anembodiment of the present invention;

FIG. 11 is a perspective view of a PCB-based microactuator in accordancewith an embodiment of the present invention;

FIGS. 12 and 13 are illustrations showing electrowetting chips inaccordance with an embodiment of the present invention;

FIG. 14 is graph of the MALDI-TOF MS spectrum of oils depicting the %intensity as a function of mass;

FIG. 15 is an illustration of a droplet microactuator stamping platformin accordance with an embodiment of the present invention;

FIG. 16 is an illustration of time lapsed images of a stampingexperiment in accordance with an embodiment of the present invention;and

FIGS. 17 and 18 are graphs of the MALDI-MS for a blank droplet aftercross-contamination experiments and depicting the % intensity as afunction of mass.

8 DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to droplet-based protein crystallizationinvolving a screening chip for varying protein crystallizationconditions and an optimization chip which can be used to set up multipleconditions around a screening condition to identify optimal conditionsfor crystallization. The present invention also relates to adroplet-based multiwell plate involving integrated fluid handlingcapability, such as in a planar multiwell plate format. The presentinvention further relates to a droplet-microactuator stamping platforminvolving devices, systems and methods for stamping samples on asubstrate. Further details of these aspects, as well as dropletmicroactuator architecture and operations, systems, kits, and otheraspects of the present invention follows hereinbelow.

8.1 Droplet-Based Protein Crystallization Overview

One aspect of the present invention provides a chip for identificationof crystallization conditions for target molecules, such as proteins andpeptides (for convenience, both are referred to herein as “proteins”).The chip is also useful for determining crystallization conditions forsmall molecules, such as small drug molecules. The chips of theinvention dramatically reduce the precious protein sample requirementsfor crystallization screening. Small sample volumes enable rapidsynthesis due to shortened crystallization times and also exploration ofa larger crystallization parameter space. Miniaturization, automation,and integration provide for simpler and more reproducible experiments,while eliminating tedious tasks and errors. Time spent by researchers insetting up screening conditions is dramatically reduced, enabling muchhigher throughput as compared to conventional methods.

In some embodiments, the system provides separate screening andoptimization chips or separate screening and optimization regions on asingle chip. The screening chip or region may, for example, be used toprovide a loading and packaging scheme to pre-fill coarse grid reagentsin all the on-chip wells or reservoirs. The optimization chip or regionmay, for example, be used to provide a loading and packaging schemewhich automatically constitutes a fine grid of conditions around auser-defined screening “lead.” The chips can provide on-chip dilutionsfrom stock solutions of the reagent constituents (e.g., salt, pH buffer,precipitant, and water). FIG. 1 provides a schematic view of a 96-welloptimization chip 100 that automatically sets up 96 fine grids fromuser-filled reservoirs. Multiple fluid ports and/or reservoirs may beprovided, such as for salt 112, precipitant 114, pH buffer 116, water118, protein 122, additive 124, and waste 126.

In one embodiment, the chip is provided as a component of a systemgenerally including a microprocessor, one or more data output componentsand one or more data input components. In the operation of such asystem, one or more dispensing reservoirs or wells of the chip may beloaded with one or more protein solutions. Dispensing wells may also beloaded with crystallization reagents, preferably 2, 3, 4, 5 or morecrystallization reagents, which can be combined in the mixing wells toprovide an array of crystallization conditions. The system is programmedto execute droplet operations, such as dispensing one or more sampledroplets from the protein solution; transporting one or more of suchsample droplets to a mixing well; dispensing one or more reagentdroplets from the crystallization reagents; transporting one or more ofsuch reagent droplets to at least a portion of the mixing wells to yieldan array of wells comprising crystallization droplets, said arrayrepresenting multiple crystallization conditions; and combining one ormore sample droplets with each crystallization condition to potentiallyyield one or more crystallization droplets comprising one or moreprotein crystals. It is understood in alternative embodiments that themixing may take place on a chip but not necessarily in a well.

Similarly, the chip may include one or more dispensing wells withprotein solution loaded therein and one or more mixing wells comprisingcrystallization droplets loaded therein, said crystallization dropletsrepresenting multiple crystallization conditions. The system may beprogrammed to execute steps, such as dispensing one or more sampledroplets from the protein solution and transporting at least a portionof the dispensed sample droplets to the mixing wells. Typically, atleast a portion of the mixing wells each receives one or more of thesample droplets, thereby yielding an array of crystallization reagentscomprising protein sample and having the capacity to one or more proteincrystals.

A technique was surprisingly discovered which permits droplets with highconcentrations of protein to be manipulated. In one embodiment, theprotein solution has a concentration which exceeds about 1 mg/mL, about10 mg/mL, about 50 mg/mL, or about 100 mg/mL. Further, the system of theinvention can operate using exceptionally low volumes of proteinsolution. For example, in some embodiments, the amount of proteinsolution required for each crystallization condition tested does notexceed about 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nL.

Importantly, small protein droplet volumes mean faster equilibration orcrystal formation. Crystals formed in small drops may perform betterthan larger ones in diffraction studies because they have the advantageof uniform and rapid freezing when transferred into liquid nitrogen.Liquids can be dispensed, transported, and mixed on-chip without theneed for robotics. The system can be self-contained, which results inhigher quality of data due to consistency of the setup. Instrumentmanufacturing is much less costly than conventional robotic approaches,and disposable chips can be provided which are relatively inexpensive.Cost per screening condition is extremely low compared to conventionalmethods. Moreover, as the number of wells on the same chip goes up, costper condition is decreased. For direct translation of crystallizationhits to larger scale, droplet volumes should be accurate and precise andin the current invention, the dispensed droplet volume is much moreprecise as compared to conventional methods, e.g., <2%. Dropletoperations and pathways are selected through a software control panel toempower users to readily create any combination of optimizationconditions. The flat geometry provides better visualization of crystalsand enables image analysis of droplets due to uniform illumination. Theinvention is compatible with Society for Biomolecular Screening (SBS)multiwell plate footprint and well-to-well pitch for any furtherautomation steps.

The chips of the invention are fabricated using a scalable architecture,which permits a large number of on-chip crystallization conditions. Forexample, the number of conditions can be greater than 50, 100, 500,1000, 5000, 10,000, 50,000, 100,000, 500,000, or 1,000,000. Theinvention also provides a programmable droplet-based microfluidic wellplatform, e.g., a platform having greater than 50, 100, 500, 1000, 5000,10,000, 50,000, 100,000, 500,000, or 1,000,000. The system of theinvention can also include droplet control software and instrumentation.

In some embodiments, the chips can accomplish all steps of synthesis(e.g., making use of a variety of droplet operations) and analysis,including for example, sampling, sample preparation, sample-processing,mixing, incubation, detection, and/or waste handling. These steps can behandled on-chip without requiring a significant off-chip support system.In some embodiments, the invention can specifically excludecontinuous-flow or droplet-based flow in fixed-channel-basedmicrofluidics.

The invention is preferably provided as a bench-top instrument or as ahandheld instrument. In one embodiment, discrete nanoliter-sized orsub-microliter-sized droplets are directly transported or manipulatedusing a microactuator structure described hereinbelow with reference toSection 8.8. In addition to transport, other microfluidic operationssuitable for use in the invention include merging, splitting, mixing anddispensing of nanodroplets by varying the patterns of voltageactivation. Large numbers of droplets can be simultaneously andindependently manipulated allowing complex protocols to be flexiblyimplemented directly through software control. There is no joule heatingor electrophoresis within the droplets and near 100% utilization ofsample or reagent is possible because no priming is performed.

Further, the invention enables the splitting, dispensing, and/ortransport of protein solutions having unprecedented high concentrations.For example, the invention includes chips, systems and methods ofsplitting, dispensing, and/or transporting protein solutions havingconcentrations that exceed than about 1, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 mg/mL. Further, in a preferred embodiment, the splitting isfacilitated by doping the silicone oil with a surfactant. For example,the surfactant may be a lipophilic surfactant, such as Triton X-15.Other suitable filler fluids and surfactants are described hereinbelowwith reference to Section 8.8.4.

Nucleation and growth phases can be separated by performing on-chipdilutions after nucleation is observed to favor growth and stop furthernucleation, as such a dilution method was reported to yield 6× thenumber of crystals suitable for X-ray diffraction compared toconventional methods. Crystal harvesting can be automated by transportof crystal-laden droplets for automatic loading into capillaries forX-ray diffraction studies. The crystals can, for example, be loaded bycapillary action. Alternatively, crystals can be loaded by activevertical axis actuation through electrostatics.

In one embodiment, the screening chip includes a multiwell plate (alsoreferred to as a microtiter plate). The multiwell plate may bepre-loaded with crystallization reagents. The plate can also include aport for introduction of protein solution, and microfluidics for movingdroplets of protein solution into and between wells of the plate. Thechip can be provided as a component of a system programmed to separatethe protein solution sample into multiple droplets, each of which ismoved to a preloaded well where it is combined with the preloadedcrystallization reagents. The contents of the wells or other regions ofthe chip can then be analyzed automatically or manually for the presenceof crystals. In some embodiments, the dimensions may be set up toconform to the SBS footprint dimensions for comparable microplates,e.g., for a 96, 384 or 1536-well plate.

In another embodiment, the optimization chip includes multiwell platewith one or more access ports for inputting reagents, as well as one ormore access ports for inputting protein solution. The multiwell platemay be pre-loaded with reagents. Examples of useful reagents include pHbuffers, salts, precipitants and other crystallization reagents. Theplate can also include a port for introduction of protein solution. Asystem including the chip can be programmed to effect droplet operationsto separate the protein solution sample into multiple droplets, each ofwhich is moved to a well or processing region, and to separate thereagent solutions into multiple droplets, each of which is moved to awell or processing region. The system can be programmed to permit theuser to control mixing and dilution of reagents and protein sample so asto systematically vary conditions in each well according to parametersset by the user. Ideally, these parameters are established based onprior analysis using a screening chip, as described above.Alternatively, results from a screening chip are used by the system toautomatically select parameter variables for use in the optimizationchip. The contents of the wells can then be analyzed automatically ormanually for the presence of crystals. In some embodiments, the chipdimensions may conform to the SBS footprint dimensions for comparablemicroplates, e.g., for a 96, 384 or 1586-well plate. While there are nostandards yet on higher number of wells in a microtiter plate, thetechnology of the invention enables scaling down to accommodatethousands of wells in the same SBS footprint.

8.2 Droplet-Based Protein Crystallization Examples

The following non-limiting examples are provided only for the purpose ofillustrating various aspects of the invention and should not beconstrued as limiting the scope of the invention.

8.2.1 Microfluidic Platform Development & Material Compatibility

A prototype chip with sample injection elements, reservoirs, dropletformation structures, fluidic pathways and mixing areas, was fabricatedto test the various components of the microfluidic platform. Droplets(25 nL) of several model proteins (lysozyme, glucose isomerase,proteinase K, grp94, hsp90, and α-haemolysin) were reliably dispensedand manipulated on-chip using electrical fields. The proteinconcentrations were in range of tens of mg/mL which is typical forprotein crystallization experiments. To manipulate this highconcentration of proteins, the filler fluid (silicone oil) was modifiedwith an oil-soluble surfactant. Crystallization reagents containingcommonly used precipitants, salts, and buffers were also successfullydispensed and transported on-chip.

8.2.1.1 Microfluidic Platform Development

A programmable droplet-based chip was fabricated. The fully assembledchip also included a transparent top-plate containing access holesaligned to the loading ports. The overall chip size was 25 mm×50 mm with97 discrete electrodes at a 500 μm pitch. The nominal droplet volume was˜25 nL.

8.2.1.2 Dispensing and Transport of Proteins

Proteins tend to non-specifically adsorb to surfaces they come incontact with, especially hydrophobic ones. In addition to contamination,protein adsorption also renders surfaces permanently hydrophilic. Thisis detrimental to droplet manipulation since the chip surface cannot beswitched back to being a hydrophobic surface by electric fields.Therefore any contact between a liquid droplet containing proteins andthe chip surfaces should be avoided to prevent contamination andfacilitate transport. The use of a low surface tension oil such assilicone oil helps in the transport of protein solutions on our chips.The oil forms a thin film between the droplet and the surfaces, therebyminimizing adsorption and contamination. However, the stability of theoil film decreases with increasing protein concentration (which lowersthe interfacial tension with oil) and consequently droplets havinghigher protein content are expected to be more difficult to manipulateon-chip.

Crystallization experiments sometimes require high concentrations (tensof mg/mL) of protein to create super-saturation conditions. Means forperforming operations, such as transporting, dispensing, and splitting,on high concentration of proteins with electric field-based manipulationon the chip were surprisingly discovered. The droplet formation process(using only electric fields) is more severely affected than transportdue to protein adsorption, since the liquid in the reservoir has a muchlarger surface area to adsorb to than a unit droplet. For example,though 10 mg/mL BSA droplets (manually dispensed) were transportable,droplet formation worked only for solutions with less than 0.01 mg/mLBSA. Though it is possible to use external pressure sources to assistthe droplet formation for high protein concentration solutions, it addsto the mechanical complexity of the system. It is therefore preferableto find a solution that would enable dispensing of high concentrationsof proteins using only electric fields.

The on-chip compatibility of several model proteins including lysozyme,proteinase K, glucose isomerase, grp94, hsp90, and α-haemolysin weretested. A low viscosity silicone oil (<2 cSt) was used as the fillerfluid in all experiments. 0.1% (w/w) Triton X-15, a lipophilicsurfactant, was added to the oil so that high concentrations proteindroplets could be formed or dispensed from on-chip reservoirs. Thesurfactant lowered the surface tension of the oil and increased thestability of the oil film between the droplet and the chip surfaceswhich significantly reduced adsorption.

Using the surfactant modified silicone oil, fully automated dropletformation was possible up to protein concentrations of 10's mg/mL usingvoltages less than 50V (which is typically used for simpler liquids suchas water). This is 3 orders of concentration improvement over theresults obtained using unmodified silicone oil. The volume of each unitdroplet was ˜25 nL. Table 1 lists the proteins with the highestconcentration tested and found to be most compatible with the system ofthe present invention. TABLE 1 Protein Compatibility Chart ProteinConcentration Lysozyme 75 mg/mL Glucose Isomerase 30 mg/mL Proteinase K20 mg/mL Grp94 27 mg/mL Hsp90 30 mg/mL α-haemolysin 10 mg/mL

8.2.1.3 Dispensing and Transport of Crystallization Reagents

Crystallization reagents usually consist of three components—a salt,precipitant and a buffer. Commonly used precipitants include inorganicsalts, organic polymers such as polyethylene glycols (PEG), non-volatileorganics such as 2-methyl-2,4-pentanediol (MPD) and volatile organicssuch as isopropanol. The individual components used in crystallizationreagents span a wide range of physical and chemical properties, and itis important to evaluate their compatibility with the system. Saltconcentrations can be as high as several moles per liter.

The pH of buffers spans a large range from acidic (pH 3.0) to basic (pH8.0). High molecular weight PEGs have viscosities as high as 100 cP fora 50% (w/w) solution in water. Organics such as isopropanol have a lowinterfacial tension and may also partition into the oil. Surfactants(which lower interfacial tension) are also used in the crystallizationof membrane proteins.

To demonstrate dispensing and transport of crystallization reagentson-chip, a representative subset of Hampton Research's sparse-matrix,Crystal Screen™, was chosen and which covered a range of the salts,buffers and precipitants. Table 2 lists the Crystal Screen™ reagentsused in the compatibility experiments. Solutions containing 0.001%,0.01% and 0.1% of Triton X-100 surfactant (v/v) were prepared inphosphate buffer for studying the effect of interfacial tension ontransport. The interfacial tension of the surfactant solution with oilvaried between 7 mJ/m² (0.1% Triton X-100) to 33 mJ/m² (0% TritonX-100). TABLE 2 Crystal Screen ™ reagents tested on-chip and theircomponents Reagent Salt Buffer Precipitant 1 0.02M Calcium chloride 0.1MSodium acetate, 30% v/v 2-methyl-2,4- pH 4.6 pentanediol 2 0.4MPotassium sodium tartrate 3 0.4M Ammonium dihydrogen phosphate 4 0.1MTris 2M Ammonium sulfate hydrochloride, pH 8.5 5 0.2M tri-Sodium citrate0.1M Sodium HEPES, 30% v/v 2-methyl-2,4- pH 7.5 pentanediol 6 0.2Mmagnesium 0.1M Tris 30% w/v polyethylene chloride hydrochloride, pH 8.5glycol 4000 7 0.1M sodium 1.4M sodium acetate cacodylate, pH 6.5trihydrate 8 0.2M tri-sodium citrate 0.1M sodium 30% v/v isopropanolcacodylate, pH 6.5 9 0.2M ammonium acetate 0.1M tri-sodium citrate, 30%w/v polyethylene pH 5.6 glycol 4000 13 0.2M tri-Sodium citrate 0.1M Tris30% v/v polyethylene hydrochloride, pH 8.5 glycol 400 15 0.2M ammoniumsulfate 0.1M sodium 30% w/v polyethylene cacodylate, pH 6.5 glycol 8000

FIG. 2 plots the maximum droplet transfer frequency as a function ofvoltage for different surfactant solutions. The plot indicates thatdroplets with lower interfacial tension require less voltage to betransferred at a particular rate. However, the drop in voltagerequirement is not significant and is only 10V for transfer at 12 Hz.Droplet dispensing and transport worked for all the crystallizationreagents tested. Table 2 above lists the individual salts, buffers andprecipitants which constituted the tested reagents. Though 100%isopropanol is completely miscible with silicone oil, mixtures ofisopropanol and water up to 80% isopropanol (v/v) were immiscible insilicone oil. Droplet manipulation using electric fields is independentof salt concentration from 1 μM to 2M. The above results indicate thatliquids with a wide range of properties are compatible with the systemof the present invention.

8.2.2 Microbatch Protein Crystallization on the Bench

The effect of oils with varying viscosities and volatility oncrystallization was tested on the bench using a crystal screen for henegg white lysozyme. The oils tested were 1 cSt, 1.5 cSt and 2 cStsilicone oil, paraffin oil, and fluorosilicone oil. Of all the oilstested, the 2 cSt silicone oil had the right combination of lowviscosity preferable for electric field mediated manipulations and lowvolatility required for long crystallization experiments, and thereforewas used for the on-chip crystallization experiments. The addition ofsurfactants to the oil also did not affect the crystal formationprocess. Screening experiments were also performed on the bench forglucose isomerase and proteinase K to identify crystallizationconditions that were subsequently tested on-chip.

8.2.2.1 Effect of Oils on Crystallization

Silicone oils, paraffin oils, or a mixture of the two are the mostcommonly used oils in microbatch-in-oil crystallization. A combinationof fluorosilicone oil (dense) and paraffin oil (light) has also beenused in a specific case of microbatch referred to as the floating-dropmethod.

1 cSt silicone oil, 1.5 cSt silicone oil, 2 cSt silicone oil, paraffinoil and a fluorosilicone oil were tested as potential candidates foron-chip microbatch-in-oil crystallization. A crystal screen for lysozymewas used to evaluate the oils on the bench. 75 mg/mL lysozyme wasprepared in 0.1M sodium acetate buffer, pH 4.6. Twelve differentconcentrations of NaCl in water (1.2M to 3.0M in steps of 0.2M) wereused as the precipitant. The screens were set up in 96-well plates. Atfirst, 100 μL of the oil was dispensed into the wells, followed by 5 μLof the 75 mg/mL lysozyme sample. 5 μL of the different precipitantsolutions were then added gently to the sample and allowed to mixwithout any stirring. A floating-drop microbatch experiment was set upin a similar fashion using fluorosilicone oil as the heavier oil andparaffin oil as the lighter oil. The wells were closed with a lid butnot completely sealed. The wells were inspected after 1 day, 2 days, 3days and 1 week.

Basic droplet manipulation experiments were also performed on-chip usingparaffin oil and fluorosilicone oil to evaluate the compatibility of theoil with the platform. Low viscosity silicone oils (<2 cSt) have alsoalready been shown to be compatible with the microfluidic platform ofthe present invention.

With the exception of 1 cSt silicone oil, all the other experiments gavesimilar crystals (size, shape and number) in wells with 1.2-2.2M NaCl. Amixture of crystals and precipitate was seen in wells with 2.4-2.6MNaCl. The proteins completely precipitated out in wells with higher NaClconcentration. 1 cSt silicone oil was 100% volatile and evaporatedwithin 1 day of setting up the experiment, causing the protein solutionto dry out. 1.5 cSt silicone oil also evaporated over the course ofseveral days. 1 cSt and 1.5 cSt silicone oil can still be used foron-chip crystallization if the system is completely sealed. 2 cStsilicone oil was non-volatile, but the crystals still dried out after2-3 weeks due to the high water vapor permeability of silicone oils.

The high viscosity of paraffin oil required substantially highervoltages for droplet manipulation on-chip. The fluorosilicone oildissolved the hydrophobic coating and was therefore not suitable for useon these chips. Of all the oils tested, 2 cSt silicone had the bestcombination of low viscosity and low volatility for use on-chip. Eventhough the paraffin oil/silicone oil mixture is compatible with thechips, pure silicone oils are preferred. Table 3 summarizes the variousresults obtained from the experiments described above. TABLE 3 Fillerfluid suitability for on-chip crystallization On-chip Overall Oil Benchexperiments compatibility compatibility 1 cSt silicone 100% volatile.All the oil Compatible Compatible oil evaporates in less than a day (ina sealed system) 1.5 cSt Oil evaporates within a few days. CompatibleCompatible silicone oil (in a sealed system) 2 cSt silicone oilNon-volatile. Fully Compatible Fully compatible Compatible Paraffin oilNon-volatile. Fully High viscosity Partially compatible requiredcompatible substantially higher voltages for droplet manipulation on-chip

8.2.2.2 Protein Crystallization on the Bench

Protein crystallization screens were set up for three modelproteins—lysozyme, proteinase K and glucose isomerase. 2 cSt siliconeoil with 0.1% Triton X-15 was used as the oil medium to simulateconditions similar to those on chip. The screens were set up in 96-wellplates. 100 μL of the oil was first dispensed into the wells followed by5 μL of the protein sample. 5 μL of the precipitant solutions was thenadded gently to the sample and allowed to mix without any stirring.

Lysozyme—75 mg/mL lysozyme was prepared in 0.1M sodium acetate buffer,pH 4.5. A concentration gradient of sodium chloride in water (1.2M to3.0M) was used as the precipitant. Table 4 summarizes the results of thelysozyme screen. TABLE 4 Lysozyme screen results Precipitant (NaCl)After 2 days 1.2M Crystals 1.4M Crystals 1.6M Crystals 1.8M Crystals2.0M Crystals 2.2M Crystals 2.4M Precipitate and crystals 2.6MPrecipitate and crystals 2.8M Precipitate 3.0M Precipitate 3.2MPrecipitate

Proteinase K—20 mg/mL proteinase K was prepared in 25 mM HEPES buffer,pH 7.0. Reagents from Hampton's Crystal Screen™ (indicated in Table 5)were used as the precipitant. Table 5 summarizes the results of theproteinase K screen. TABLE 5 Proteinase K screen results Precipitant(Crystal Screen ™) After 2 days Reagent 6 Crystals Reagent 12 ClearReagent 17 Crystals Reagent 19 Clear Reagent 22 Crystals Reagent 28Crystals Reagent 30 Crystals Reagent 31 Crystals Reagent 36 CrystalsReagent 39 Crystals Reagent 40 Crystals Reagent 41 Crystals Reagent 42Crystals Reagent 43 Clear Reagent 48 Crystals

Glucose isomerase—30 mg/mL glucose isomerase was prepared in deionizedwater. Reagents from Hampton's Crystal Screen™ (indicated in Table 6)were used as the precipitant. Table 6 summarizes the results of theglucose isomerase screen. TABLE 6 Glucose isomerase screen Precipitant(Crystal Screen ™) After 2 days Reagent 9 Crystals Reagent 10 CrystalsReagent 14 Crystals Reagent 15 Crystals Reagent 18 Precipitate Reagent21 Clear Reagent 23 Crystals Reagent 24 Precipitate Reagent 32Precipitate Reagent 35 Precipitate Reagent 44 Clear

The presence of 0.1% Triton X-15 surfactant in the oil did not affectcrystallization on the bench.

8.2.3 Protein Crystallization On-Chip & System Integration

Lysozyme, proteinase K and glucose isomerase were crystallized on-chipusing the surfactant-modified 2 cSt silicone oil as the filler fluid.Droplets of the protein and precipitant were automatically dispensed,mixed, and incubated on-chip. The precipitant was chosen from theresults of the screening experiments on the bench. Crystals typicallyappeared after 1 day and the shape and size of the crystals were similarto those obtained on the bench. A coarse crystallization screen was alsoset up on-chip for lysozyme varying the precipitant (NaCl)concentration. Four different precipitant concentrations wereautomatically generated on-chip by dilution (3:0, 2:1, 1:2, 0:3) andmixed with a 25 nL lysozyme droplet. The results compared favorably withexperiments on the bench.

8.2.3.1 Protein Crystallization On-Chip

Three model proteins, lysozyme, proteinase K and glucose isomerase werecrystallized on-chip using 2 cSt silicone oil with 0.1% Triton X-15 asthe filler fluid. Protein concentrations were identical to those used onthe bench. 1.8M sodium chloride in water was used as the precipitant forlysozyme. Crystal Screen™ Reagent 6 (0.2M MgCl₂, 0.1M Tris HCl, pH 8.5,30% w/v PEG 4000) was used as the precipitant for proteinase K, andCrystal Screen™ Reagent 14 (0.2M CaCl₂, 0.1M HEPES-Na, pH 7.5, 28% v/vPEG 400) was used as the precipitant for glucose isomerase. 25 nLdroplets of the protein and the crystallization reagent wereautomatically dispensed, mixed, and incubated on-chip.

Crystals appeared after 1 day for all the three proteins tested. Thecrystal size and shapes were similar to those obtained on the bench.FIG. 3 shows lysozyme crystals formed on-chip at 20× magnification. Thesize of the crystals was ˜140 μm. FIG. 4 shows crystals of glucoseisomerase on-chip at 20× magnification and FIG. 5 shows crystals ofproteinase K at a 40× magnification. The glucose isomerase crystals were˜70 μm and the proteinase K crystals were ˜50 μm.

8.2.3.2 Coarse Matrix Screen for Lysozyme

As a demonstration and integration of all the functions, the experimentintegrated dispensing, transport, and mixing of a lysozyme droplet with4 different concentrations of a precipitant (NaCl) to create a screen onthe chip. The lysozyme sample (75 mg/mL) and two differentconcentrations of NaCl (1.2M and 3.0M) were injected into on-chipreservoirs. Intermediate concentrations of 1.8M and 2.4M NaCl wereautomatically generated on-chip by dispensing and mixing 2 droplets of1.2M NaCl with 1 droplet of 3.0M NaCl and 1 droplet of 1.2M NaCl and 2droplets of 3.0M NaCl respectively. One ˜25 nL droplet of 75 mg/mLlysozyme was then dispensed and mixed with 3 droplets each of the 1.2M,1.8M, 2.4M and 3.0M NaCl solutions. A similar experiment was set up onthe bench as a control.

Crystals were observed using 1.2M and 1.8M NaCl both on-chip and on thebench after a day. Images of the results obtained for the differentprecipitant conditions on-chip are shown in FIGS. 6A-6D. This experimentdemonstrated two important aspects of the protein crystallizationprocess. The first aspect relates to fluidic operations involved in thesetup of coarse screen. Once the samples and reagents are loaded, acomprehensive screening experiment can be set up using a very smallquantity of protein without any manual intervention. For example, 4different conditions were set up using only 100 nL of protein. Thesecond aspect relates to automatic dilution of precipitants foroptimization. It was shown that using two different concentrations ofthe precipitant, intermediate concentrations can be generatedautomatically on-chip. This forms the basis of an optimizationexperiment in which a matrix of conditions is usually set up around a“lead” seen in the coarse screen.

Among other things, the work described herein demonstrates ananodroplet-handling platform for manipulating a wide variety ofproteins and reagents, the compatibility of crystal nucleation andformation with electric field mediated droplet operations, favorabletranslation between on-chip nano-batch and off-chip micro-batch, andscreening of multiple conditions and on-chip setup of dilutions foroptimization.

8.3 Droplet-Based Protein Crystallization Further Examples

Further examples relevant to the design, development, fabrication, andvalidation of a digital microfluidic protein crystallization chip areincluded hereinbelow and include a screening chip which requires onlyone drop of protein (4 μL) for screening with 384 pre-filled reagentsand an optimization chip which requires 1 μL of protein for fine gridoptimization with 96 reagents constituted fully on-chip from theindividual components of a screening reagent that yielded a lead.

Separation of screening and optimization chips. The requirements forscreening and optimization are different, so two different chips can beused, even though the digital microfluidic multiwell chip can serve asthe basic platform for each. Alternatively, a single chip with twodifferent regions (screening and optimization) can be used. In thescreening phase, researchers typically screen a protein with a known setof reagents where the concentrations of the constituents are pre-set ina coarse grid covering a broad range of crystallization space.Therefore, the screening chips and/or cartridges including the screeningchips can be pre-loaded with known reagents saving the user from havingto load 384 wells. In the optimization phase, however, researchers picka coarse grid condition that yielded a lead during screening and thenprepare a fine grid of reagent concentrations by performing a number ofdilutions. Therefore, the optimization chip can set up 96 conditionsthrough on-chip dilutions from a few reservoirs filled with the stocksolutions of the constituents. The dimensions of both the chips canconform to the SBS standard multiwell plate footprints (85.48×127.76mm²) and well-to-well pitch (9/4.5/2.25 mm for 96/384/1536 respectively)so that the chips are readily compatible with robotic plate-handlingequipment.

Only about half of all proteins crystallized, using any method, haveturned out to be useful in determining the 3D structure. The reason isthat the “leads” obtained during screening may not form large crystalsin the optimization phase. Vapor diffusion (VD) and free interfacediffusion (FID) are particularly challenging to optimize because a largecrystallization space is sampled in the process, which is advantageous,but the exact conditions for crystallization are not known.

A microbatch method lends itself readily for translation from lowvolumes on-chip to larger volumes on the bench if the volume of thereagent and the protein are known accurately. The precision of dropletdispensing on our platform allows for accurate translation of optimizedcrystallization conditions from nanoliter-level on-chip tomicroliter-level on the bench.

8.3.1 Digital Microfluidic Multiwell Plate

A scalable architecture can be used to make a chip with 384 on-chipcrystallization conditions. A programmable droplet-based microfluidic384-well platform can also be made, along with relevant droplet controlsoftware and instrumentation. The key fluid handling capabilities forprotein crystallization was demonstrated and the protein required foreach screening condition can further be scaled down from 25 nL tosub-nanoliters.

8.3.1.1 Screening and Optimization on Digital Microfluidic Chips

Screening-Chip Architecture

An important issue in the design of the screening chip is the routingcomplexity of the electrical signals that control the well-plateelectrodes. To reduce the product cost and simplify design, it is usefulto minimize the number of electrodes needed to provide droplet pathwaysfrom the protein reservoir to the screening wells.

Algorithms for fault tolerance can also be developed to get around theunlikely event of a protein droplet getting stuck on a pathway. Eachprotein sample loaded into a reservoir is very precious so instead ofthrowing away the chip because of one fault, the algorithm canadaptively find a path for other droplets that avoids the fault byconfiguring multiple mutually-disjoint paths from a designated reservoirto the well.

Optimization Chip Architecture

From FIG. 7, it can be seen that the microfluidic chip 700 design ofthis specific embodiment of the invention preferably has 77 electrodes702 to transfer droplets 704 between four wells 706 (see also FIGS. 8and 10). To independently control all 384 wells, it would require 1000'sof input/output electrical pads which becomes very expensive. As such, a4-“pin-limited” design was implemented where every fourth electrode isconnected together to perform droplet operations between wells.

In pin-limited designs, the simultaneous movement of multiple dropletsis restricted where two or more moving droplets might get inadvertentlycoupled. A method can be developed to analyze interferences betweendroplets in every clock cycle. In some steps of the fine grid setupprocess, interference may be inevitable, then one droplet may have toundergo a stall cycle (i.e., stay in its current location). For both 96and 384 well-plate arrays, the minimum number of independent controlpins sufficient to provide full control can be identified (especiallydesirable for concurrently executing multiple steps during fine gridsetup) of a single droplet. For any electrode in the array, the controlpins for all its adjacent electrodes are preferably distinct. Pinlayouts can be identified, i.e., assignments of control signals toelectrodes, such that maximum freedom of movement of groups of dropletsin the array can be ensured.

For the optimization chip, droplet routing can be managed betweenloading reservoirs and wells as well as between intermediate reservoirsand wells. Droplet routes can be identified with minimum lengths, whereroute length is measured by the number of electrodes in the path fromthe starting point to the destination. During droplet routing, it ispreferable for a minimum spacing between droplets to be maintained toprevent accidental mixing, except when droplet merging is desired. Formultiple droplet routes that may intersect or overlap with each other,fluidic constraint rules must be introduced to avoid undesirablebehavior.

Algorithms can be used to schedule droplet operations based on optimalutilization of the stock solutions without requiring users to refill thestock solutions. State-of-the-art in computer architecture can beleveraged to build a microfluidic “compiler” for the optimization chipas there are a number of similarities between the fine grid setupproblem and the problem of compiling a program written in a high-levelprogramming language (such as C/C++). The compiler typically decomposesuser-level code to a series of hardware instructions based on theinstruction-set architecture of the underlying microprocessor. Next, thecompiler determines parallelism between instructions, maps theseinstructions to functional units, allocates the hardware registers forinstruction execution, and performs instruction scheduling. Themicrofluidic compiler can view the given stock solutions as the initialvalues of variables in the user program and fine grid conditions canserve as the final values of another set of variables. The latter set ofvariables can be controlled by manipulating the initial set of variablesas well as additional intermediate variables. Once the compilerdetermines the set of microfluidic operations required for fine gridsetup, a graph model can again be used to describe the fine grid setupprotocol, where the vertex set is in one-to-one correspondence with theset of operations and the edge set represents dependencies between theoperations. Next, a synthesis tool can be developed to generate detailedimplementations from the sequencing graph model.

The proposed synthesis tool can perform both architectural-levelsynthesis (e.g., scheduling and resource binding) and geometry-levelsynthesis (e.g., layout of module placement and routing) for a fine gridsetup based on user-defined concentrations. The output of the synthesisprocess can include a mapping of operation to wells, a schedule for thevarious operations, and the placement of the modules. The synthesisprocedure can identify a desirable design point that satisfies the inputspecifications and also optimizes some figures of merit, such as thevolumes of the stock solutions and number of wells for fine grid setup.All these steps can be transparent to the user.

8.3.1.2 Design and Fabrication of Digital Microfluidic Chips

Dispensing reservoirs and transport electrodes can be made so thatdroplet volume (determined by the electrode area and gap height) can bescaled down from 25 nL (500×500×100 μm³) to 10.5 nL (375×375×75 μm³),e.g., as shown in FIGS. 8 and 9. The number of wells can, for example,be 24, 96, 384, or even 1536. Referring to FIG. 8, a screening chippre-filled with 384 reagents for crystallization is shown. Overalldimensions conform to SBS multiwell plate. The 96-well modules 800 can,for example, have input ports 802 and a dispensing reservoir 804 with asample loading port designed to accept about 1 μL of protein. Otherdispensing reservoirs can be provided. The zoom-in view depicts, as anexample, protein and reagent sample mixes 808, pre-loaded reagents 812,and a protein droplet 814 en route. A combination of 4 such 96-wellmodules yields a 384-well plate and a further combination of 16 yields a1536-well plate. The pitch 906 in each of the 96-well modules is that ofa 1536-well plate i.e., 2.25 mm which can accommodate a 6×6 pattern of375×375 μm² electrodes. The line spacing between the electrodes can be˜1 mil (25.4 μm), the gap height formed by a photopatternable gasketcan, for example, can be about 3 mils (76.2 μm), and multiple layers canbe used to perform electrical routing. The digital microfluidic chipscan be fabricated in printed circuit board (PCB) processes that havebeen adapted to various fabrication needs. PCB processes allow themanufacture of the chips inexpensively and therefore enable widespreadadoption.

FIG. 9 schematically depicts an optimization chip that automaticallysets up 96 fine grid conditions. 96 optimization wells in 1536-pitchoccupy only 1/16th area. The zoom-in depicts reagents, appropriatelydiluted on-chip, transported to a well. For example, pH buffer droplet A902, precipitant droplet B 904, and salt droplet C 906 are shown beingtransported.

Further description of the architecture and operation of a typicaldroplet microactuator capable of being used with this aspect can befound hereinbelow with reference to Section 8.8.

8.3.2 Reagent Loading & Pre-Processing On-Chip

Separate screening and optimization chips were developed. For thescreening chip, for example, a loading and packaging scheme can bedeveloped to pre-fill coarse grid reagents in all the 384 wells. For theoptimization chip, for example, a system of the invention can beprogrammed to automatically constitute a fine grid of 96 conditions.On-chip dilutions can also be performed from stock solutions of thereagent constituents (e.g., salt, pH buffer, precipitant, and water) forthe optimization chip.

8.3.2.1 Screening Chip with 384-Wells Pre-Filled with Reagents

Protein crystallization is an empirical science that has benefited fromthe development of sparse matrix screens that coarsely sample largeregions of crystallization space. Sparse matrix of reagents, describedby Jancarik and Kim, has been successfully used as an initial screen forcrystallizing more than 1000 proteins. Typical initial screens performedin structural biology labs (for example, the Hampton Research CrystalScreens I and II) consist of 50-100 different reagents and typicallyconsume 200-400 μL of concentrated protein sample. Hampton Research alsosells about 900 additional crystal screening reagents many of which arevariants of Crystal Screens I and II. 1536 reagents can be used formicrobatch screening and the screening chips can be filled with 384reagents to save the user from having to load all the wells.

8.3.2.2 Choice of Reagents

In one embodiment, the screening chips can have 384 reagent wellson-chip. For example, five (5) different chips can be made withdifferent combinations of pre-filled reagents. One of these chips cancontain sparse matrix conditions and a few combinations of salts,PEG/salts, PEG/buffer, and other precipitants totaling 384 conditions.The remaining four (4) chips can be based on HWI's 1536 reagents, whichinclude 46 salts at 10 different concentrations (460), 3 distinct PEGsat 5 different concentrations combined with each of the 46 salts (690),eight different PEGs ranging in molecular weight from 200 to 35,000 Daat 5 concentrations combined with buffers ranging in pH from 4.8 to 10.4(226), 20-step fine screens of ammonium sulfate, lithium chloride, andpotassium thiocyanate (60), and sparse matrix based on Crystal Screens Iand II (100) adding up to a total of 1536 conditions. An example of thescreening chip is found in Table 7. TABLE 7 Digital microfluidicscreening chip specifications Protein Sample Reservoir 1 ReagentsPre-filled 384 Screening Wells 384 Protein Reservoir Capacity 400droplets Unit droplet volume ˜10 nL Total Protein Volume Required 4 μLSBS multi-well plate Footprint 88.48 × 127.76 mm² Well-to-well pitch2.25 mm

8.3.2.3 Chip Layout

The overall chip size can be that of a standard SBS multi-well plate.Thus, 384 wells can be arranged in 1536-well pitch (2.25 mm) withelectrode pathways to connect these wells to reagent and protein inputloading ports. Reagent and protein droplets can be transported alongthese pathways from their input loading ports to the wells and otherdroplet operations can be conducted. 384 wells in a 1536-well pitchoccupy only ¼^(th) area of the digital microfluidic multiwell plate. Therest of the chip real estate can be used for accommodating the reagentand protein input wells. In addition to the protein reservoir that auser loads, two additional reservoirs can be included that the user canload. These additional reservoirs can, for example, be loaded with anyuser-selected additives such as glycerol or detergents. Additives canstabilize the proteins, e.g., to improve the quality and size of proteincrystals. In another embodiment, the number of wells can be increasedon-chip, as described in more detail above.

8.3.2.4 Reagent Loading Schemes

In order to not burden the user with filling 384 wells, all the reagentscan be pre-filled and packaged in a facility so that the user only needsto introduce a drop of protein to the chip. For effective translationfrom nanobatch on-chip to microbatch off-chip, it is essential totightly control the volume of the droplets. The droplets dispensed onthe digital microfluidic platform are very precise with a CV<2% innanoliter range. This precision can be exploited in various reagentpre-filling schemes in conjunction with other loading methods. Outlinedhereinbelow are a variety of suitable loading mechanisms:

-   -   1. Electric Field Mediated Dispensing: 384 loading reservoirs        around the periphery can be loaded with respective reagents and        the chip can be programmed to dispense a 10 mL droplet from all        these wells and transport a droplet into each of the 384        screening wells. Each loading reservoir can, for example, hold        at least 100 nL. This scheme has been demonstrated for        dispensing 25 nL droplets from 4 loading reservoirs.    -   2. Pressure-Assisted Electric Field Mediated Dispensing: 12        loading ports in 96-well pitch can be placed on either side of        the chip. 12 syringe pumps, arranged in 96-well pitch, can be        arranged to first sip the reagents from a 96-deep well block of        reagents. All the 12 pumps can be interfaced to the 12 loading        ports to push the reagents and electric field can be used to        create a surface energy well to trap a droplet. These droplets        can then be transported away to the screening wells. This method        has been successfully used to dispense multiple droplets        simultaneously, and it is readily scalable to load 1536        reagents.    -   3. Multi-well Bulb Droppers: In this scheme, reagents can be        prepared in a 384-deepwell plate. An array of droppers can be        used to sip the reagents from the deepwell plate and drop the        reagents into the loading ports on-chip surrounding 384-wells.        10 nL droplets can be dispensed from these loading ports and        transported to the screening wells.    -   4. Robotic Pipetting: Robotic pipetting can deliver precise        volumes into the screening wells.

8.3.2.5 Protein Loading

The chip can be programmed to precisely dispense 10 nL droplets 384times from a single protein reservoir. This reservoir can be constructedto hold about 4 μL of protein. A polypropylene or polyethylene orpolyester or silicone or any tape can be used as a sealing film toprevent evaporation through the loading ports during incubation.Microbatch experiments typically require a higher protein concentrationcompared to vapor diffusion experiments. In 800 experiments surveyed inthe literature, 4 were set up at <2 mg/ml and 4 were set up at >300mg/ml and a majority of the rest are set up in the 5 to 10 mg/ml rangeof protein concentration. Dispensing and transport of protein dropletswith concentrations of 75 mg/ml has been demonstrated.

8.3.2.6 Optimization Chip Programmed to Set Up 96 Conditions

In this embodiment, an optimization chip is made and the system isprogrammed to set up 96 conditions from 2 reservoirs of stock solutions,2 reservoirs of buffer, and 1 reservoir of water. Sparse matrixscreening for crystallization requires a relatively predictable amountof protein and can be done in a single experiment. However, it is oftenthe refinement of the initial crystallization hit that takes the longesttime and uses the most material. Typically, upon getting a hit from asparse matrix screen, an investigator will construct a series of gridsaround the index condition. Microbatch particularly requires fine gridsfor optimization after screening. This process involves a number oflabor-intensive dilutions. It is very difficult to do this with currenttechnologies on small volumes of proteins. In addition, human pipettingis only reliable in the microliter range with about 110% volumevariability and in a very fine grid this level of variability will notprovide enough resolution between different conditions. Roboticpipetting is another option, but robots that are precise and accurate insingle-digit nanoliter dispensing are costly. Therefore, the chips ofthe invention, which can dispense with <2% CV in the nanoliter-regimeare a very cost-effective alternative to expensive fluid handlingrobots. A major advantage of digital microfluidics is the softwareprogrammability of fluidic operations due to electronic control ofliquids. Therefore, this versatile feature can be exploited byfabricating chips that perform all the dilutions and create, initially,96 fine grid conditions from 3 user-loaded constituent stock solutionsof the salts, precipitants, and buffers.

The main difference between the screening and the optimization chip isthat for the screening chip the reagents are pre-filled as theconstituents of the reagents are discrete and fixed and for theoptimization chip the reagents are constituted on-chip because theconstituents of the reagents span the entire crystallization space andvary based on the lead condition. Other than robotic pipetting, no othermicrofluidic technology offers such high levels of programmability as isrequired for on-chip dilutions.

On-chip optimization is best illustrated by an example. Hsp90crystallizes at a slightly acidic pH with PEG 4000 as the precipitantand ammonium phosphate as the salt. For example, 96 conditions were setup in a fine grid as follows: pH 4.6 and 5.6 (2 conditions), PEG 4000 insteps of 2% at 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22% (8 conditions), andammonium phosphate in factors of 2 at 50 mM, 100 mM, 200 mM, 400 mM, 800mM, and 1600 mM (6 conditions).

From the above screen, Hsp90 crystallized at pH 4.6, 10% PEG, and 0.2Mammonium phosphate. If the user loads 2.5M ammonium phosphate into oneof the on-chip stock solution reservoirs, there are a few ways ofconstituting 0.2M on-chip. In one scheme, 2 droplets of 2.5M solutioncan be mixed in an intermediate reservoir with 23 droplets of water inone step to get 0.2M solution. In this scheme, a total of 25 dropletsare dispensed and used. In another scheme where intermediate dilutionsare utilized, 2.5M would be first diluted to 1M in an intermediatereservoir by mixing 2 droplets of 2.5M solution with 3 droplets ofwater. In another intermediate reservoir, 1 droplet of 1M solution canbe mixed with 4 droplets of water to get 0.2M solution. In this scheme,only 10 droplets are dispensed and used. This example serves toillustrate that dilutions can be performed to minimize reagentconsumption for fine grid setup on-chip which minimizes the number ofdispensing steps, thereby reducing the overall error in volume.

Reservoirs can be included for HCl, NaOH, and/or other reagent buffersto constitute a full range of pH values (e.g., pH 1-7 at variousincrements) on-chip through dilutions. Among other things, theadvantages of using the envisioned chip for setting up the fine gridconditions are: low volume of protein used per condition (10 nL),automatic setup of 96 conditions inexpensively, and precision in dropletvolumes (<2% CV). An example of the optimization chip is found in Table8. TABLE 8 Digital microfluidic optimization chip specifications ProteinSample Reservoir 1 Reagents filled by user 5 Optimization Wells 96Protein Reservoir Capacity 100 droplets Unit droplet volume ˜10 nL TotalProtein Volume 1 μL Required SBS multi-well plate Footprint 88.48 ×127.76 mm² Well-to-well pitch 2.25 mm

The optimization chips can, for example, include 5 loading ports; oneeach for the stock solutions of salt and precipitant, water, and two forbuffers. Each chip can be capable of constituting 96 combinations ofreagents from these 5 reservoirs. Two (2) additional loading ports canalso be included for the optional inclusion of additives in theoptimization phase. 96 optimization wells in a 1536-well foot printoccupy 1/16^(th) of the chip area and the rest of the area can be usedfor the stock solution, intermediate, and waste reservoirs.

Fine grids can be set up on-chip in a variety of ways based on thecondition that the smallest droplet that could be generated is 10 nL. Ifthe stock solution reservoirs are large enough, large numbers ofdroplets could be combined directly with water droplets to set up anycondition. This method minimizes the number of dilutions but requiresmany dispensing steps. Another method is to constitute the desiredconcentrations by using serial dilutions (binary, decade etc). Yetanother method, as illustrated in the example, would combine both theabove methods where a stock solution would be diluted in one step towithin the proximity of required concentration and from thatintermediate reservoir fewer droplets would be directly dispensed to setup the fine grid conditions. Based on the design architecture optimizingthe layout and electrical connections, an optimum combination ofreservoirs can be chosen for direct dispensing and serial dilutions.

The goal in these experiments is to rapidly mix 2-3 droplets within areservoir. A number of schemes can be developed to perform rapid mixingwithin the reservoirs. For example, an n-electrode×n-electrode array canbe used within the reservoir to rotate a droplet to enhance mixing.

For some user-defined concentrations of stock solution, it may not beeasy to set up the required concentration range for the 8×6 conditionswith the required intervals between the concentrations. For such cases,as an alternative, the software can be designed to calculate an optimuminitial concentration of the stock solutions and prompt the user to loadthe same. Some of the additives, such as acetone, ethanol, methanoletc., may be miscible with the surrounding oil but it has been foundthat these additives do not partition into oil at the concentrations atwhich they are typically used (usually <40% w/v). However, themiscibility of any organic compounds is to be determined by the end userand an appropriate immiscible medium is to be used.

8.3.3 Protein Crystallization Screening and Optimization

In a demo of screening chip operation, 4 μL of protein can be loadedinto a screening chip using a pipettor, screen script routine run, 384protein droplets automatically dispensed and mixed up with 384pre-filled reagents.

In a demo of an optimization chip operation, the user can load 1 μL ofprotein and stock solutions of salt and precipitant, buffers, and waterusing a pipettor, input concentration range and interval for a finegrid, run fine grid script routine, inform the user via software if anymodifications to the stock solutions are required, cause the chip toconstitute 96 fine grid conditions, dispense 96 protein droplets, andmix them with the reagents. If any reservoirs do not dispense, then thechip can automatically detect and run a reload script or if any dropletgets stuck on a pathway the software can automatically detect thefailure and work around the fault. The user can incubate the chip andperiodically check for crystals.

8.3.3.1 Crystal Screening in 384-Well Digital Microfluidic Systems

The user can replicate the initial screening step of proteincrystallization using a screening chip and compare the results to thoseobtained by conventional microbatch crystallization. Several proteinscan be subjected to this screening in order to assess comparisons withtraditional microbatch screening.

The droplets in the chips can be flat and shaped as hockey pucks ratherthan as spherical droplets. This is advantageous for automated crystalscoring since image distortion due to optical aberrations would not be aconcern. An x-y micrometer stage can be adapted to fit on a standardstereo-zoom microscope and the screening chip can be viewed manually orautomatically translated to score for crystal hits within the wells byusing image processing.

PCB chips are usually opaque but can be made transparent by filling thevia-holes with a transparent epoxy if needed for visualization orpolarization microscopy. In order to eliminate the possibility offinding salt crystals, the user can run a negative control chip toidentify salt crystal formation or add a droplet of coomassie blue ormethylene blue which fill the solvent channels in protein crystals. Thisselectively colors protein crystals blue and leaves salt crystals clear.

8.3.3.2 Optimization of Crystallization Hits Using the Optimization Chip

After obtaining the screening conditions, an optimization grid typicallyhas to be set up. Within these grids, the concentration of one or two ofthe components of the sparse matrix crystallization reagent is varied.The choice of variable component and the grid size are based on theinvestigator's experience, and are limited by the amount of protein onhand, the geometry of the crystallization apparatus, and the timeallotted to the experiment. Optimization is often an iterative process,as first one grid and then another is tested until the parametersgoverning crystallization in that particular region of crystallizationspace are understood. This is, however, a strategy that is limited inits breadth. For example, even in a simple crystallization setup theremay be several components to vary, including precipitant and saltconcentration, buffer pH, additives, as well as protein concentration,in addition to environmental variables such as temperature. A standardoptimization strategy would usually first focus on constructing a gridof precipitant versus salt, wherein the precipitant concentration wouldbe varied linearly and the salt concentration varied by a multiplicativefactor (e.g. 2). After several days, the experiment would be evaluatedand additional grids set up. A much more efficient strategy foroptimization would vary as many components as possible in the sameexperiment. This becomes an attractive option especially if the samplerequirements remain modest and the labor of setting up themulti-dimensional grids can be automated. The optimization chip offersboth of these benefits, and the purpose of this experiment is todemonstrate that optimization grids can be constructed around an indexsparse matrix condition that reproduces the original crystal andparameterize its growth conditions.

For any protein, a grid of conditions can be set up around the sparsematrix condition that yielded crystals. 96 conditions can be set up,varying at least 3 components: precipitant, salt, and pH. Initially theprecipitant can be varied linearly, while the salt is varied by factorsof 2, and the pH adjusted in 1 pH unit increments. Reagent droplets canbe constituted from concentrated stock components and water on-chip, andthen mixed with an equal volume of protein in an optimization well. Forprotein-ligand co-crystallization, after formation of crystals (fore.g., Hsp90) on-chip, the user can demonstrate soaking drugs (forexample, radicicol, an antifungal antibiotic) with existing proteincrystals on the optimization chip. If the crystals do not crack orbecome damaged, then the drug-bound protein crystals can be harvested ina capillary tube for X-ray diffraction. Protein-drug co-crystallizationconditions can be similar to the protein-only crystallizationconditions. The optimization chip can be also combined with themultiwell bulb dropper dispensing scheme to load 96 drug compounds froma high-throughput screening microplate directly onto this chip. In thiscase, the chip can be programmed to set up a single condition, which isknown to give crystals, in all 96 wells. In this manner,multi-drug-single-crystal soaking studies can be performed. Thisdemonstrates a few additional uses beyond optimization for thetechnology.

Bodenstaff et al., on the other hand, observed additional crystal forms,not observed in larger volumes, in 200-500 pL of lysozyme droplets. Incases where only tiny crystals are produced on the chips, or evendifferent forms of crystals are observed, then a microfocus synchrotronbeamline can be used for the analysis of the crystals. Such beamlineshave proven to be useful for small crystal sizes in recent years. Thisbecomes feasible since the chip allows for the movement of the dropscontaining crystals into a position on-chip where the crystals could beharvested into a capillary tube.

In short, this work demonstrates manipulation and setup of hundreds ofdroplets, a high throughput 384-well screening chip, and a programmableoptimization chip for automated setup of optimization conditions throughmultiple dilutions programmed on-chip.

8.4 Droplet-Based Multiwell Plate Overview

According to another aspect, the chip of the present invention typicallyavoids the requirement for a continuous-flow approach, though chips ofthe invention may in some cases be supplemented by such an approach.Systems including chips of the invention provide flexibility andprogrammability that is comparable to robotic systems. The chips of theinvention can manipulate droplets as small as a few nanoliters involume. In certain embodiments, the chip of the invention specificallyavoids networks of microchannels, external pumps, valves, high-voltagesupplies and/or centrifugal force, though such components may beemployed to supplement certain aspects of the invention. The system ofthe invention is scalable and allows multiple liquid droplets to beprocessed in parallel. The chip of the invention can be manufactured ina highly compact form, and it is inexpensive to manufacture. A schematicexample chip 1000 is illustrated in FIG. 10 and includes a 96 well array1002, a droplet transport network of electrodes 1004 for transportingdroplets 1006, and a sample input module 1008.

8.4.1 Well Configurations

In one embodiment, the chips of the invention include a regular array ofmicroliter-volume wells or reservoirs. The chip may also include afluidic input module for loading bulk sample or reagent. A network ofdroplet transport pathways interconnects each of the wells and discretenanoliter quantities of liquid can be transported between any two wellsor inputs, e.g., to automatically and precisely generate an array ofscreening conditions. The wells and array of electrodes can be used toconduct a variety of droplet operations.

In some embodiments, wells can be physically defined by a polymer gasketmaterial and linked together by a droplet transporter network defined bythe pattern of electrodes on the chip surface. The standard pitch of thediscrete electrode elements in the network establishes a unit dropletvolume for the entire system. An open architecture in which reservoirsare defined by placement of reservoir electrodes is also possible.

In one embodiment, the chip dimensions conform to standard Society forBiomolecular Screening microplate (multiwell plate) dimensions, such asthe dimensions set forth in “ANSI/SBS 1-2004: Microplates—FootprintDimensions,” as updated on Jan. 9, 2004; “ANSI/SBS 2-2004:Microplates—Height Dimensions,” as updated on Jan. 9, 2004; “ANSI/SBS3-2004: Microplates—Bottom Outside Flange Dimensions,” as updated onJan. 9, 2004; and “ANSI/SBS 4-2004: Microplates—Well Positions,” asupdated on Jan. 9, 2004. The entire disclosure of each of thesedocuments is incorporated herein by reference for its teachingconcerning microplate standards. For example, the design can be providedin standard 96 or 384-well or 1536-well format, as well as other customformats. Use of standard well layout and spacing permits the chips ofthe invention to be compatible with conventional microplate equipment,such as pipette dispensers and read-out equipment. Standardizedembodiments will enable integration of the chips of the invention intoexisting microplate systems and workflows.

Certain designs may combine microplate standards on a single device. Forexample, one portion of the chip may conform to 96-well format forloading of samples, while another portion conforms to 384 or 1536-formatfor arraying of reactions. Other designs may divide the chip intomodules designed to perform different functions where some modulesconform to multiwell plate spacing for loading, storing or detection ofreagents or reactions while other modules may have structures designedto perform specific operations or procedures.

The chips of the invention are highly flexible and can accommodatespecialized, non-standard conformations. Further, the number of wells onthe chips of the invention can be much larger than provided for inexisting microplate specifications. For example, chips of the inventioncan incorporate greater than 1,000, 5,000, 10,000, 15,000, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or even1,000,000 sub-nanoliter reactions on a single plate.

Specifications for one embodiment of a 96 well plate are as follows inTable 9. TABLE 9 Example specifications for 96 wells on a 128 mm × 86 mmplate (chip) size. Specification Value Well pitch 9 mm Array size 12 × 8(96 wells) Transport electrode 0.75 mm width Unit droplet diameter 0.75mm Unit droplet volume 80 nL Well volume 800 nL (10 unit dropletvolumes) Plate spacing 0.15 mm Input ports 10 Input port volume 8 μL(100 unit droplet volumes) Overall chip size 128 mm × 86 mm

A relatively low-density (96-1536 wells) PCB-based platform provides abasic and inexpensive assay automation tool. For example, referring toFIG. 11, in one embodiment, the invention provides a microactuator 1100comprising a PCB-based chip 1102 that will plug into a base 1104 thatconforms to the size and shape of a standard multiwell plate. The PCBchip 1102 can have a top plate 1106, a 96-well array 1108, and a fluidinput module 1112. The base 1104 can have electrical contacts 1114 and acontroller circuit board 1116. All of the electronics required tooperate the chip 1102 can be integrated into the base 1104, e.g., withthe electrical contacts 1114 distributed on the top-side of the base1104 to make contact with the backside of the chip 1102. The entireinstrument can be powered and controlled from a standard USB port 1118or even operated as a stand-alone instrument. Power can be provided bybatteries located within the plate so that the stand-alone plateprovides completely automated fluid handling in the same form factor asa standard plate. This embodiment of an automated multiwell platform canperform many of the functions of a robotic system at a tiny fraction ofthe cost, and will be particularly useful in low and moderate throughputenvironments, including much of academic research, where the expense ofrobotic automation may be difficult to justify. Fluid handlingcapability is completely integrated within the unit which has standardplate dimensions and is compatible with conventional plate readers andhandling equipment.

Larger chips with extremely high levels of throughput and cost savingswill be useful in a variety of settings, such as drug discoveryapplications. In one embodiment, the invention is useful forhigh-throughput biological assays. For example, the chip can beprogrammed to execute on-chip dilutions and cell-handling protocols.Scaling of droplet volumes on a fully populated 128 mm×86 mm plate(chip) size at different well pitches can be seen in Table 10. TABLE 10Scaling of droplet volumes at different well pitches. Unit Unit WellWell drop Plate drop Min. Pitch Total volume diameter spacing volumefeature (mm) Rows Cols wells (nL) (μm) (μm) (nL) (μm) 9.00 12 8 96 67501500 300 675 75.0 4.50 24 16 384 844 750 150 84.4 37.5 2.25 48 32 1536105 375 75.0 10.5 18.8 1.13 96 64 6,144 13.2 188 37.5 1.32 9.38 0.563192 128 24,576 1.65 93.8 18.8 0.165 4.69 0.281 384 256 98,304 0.206 46.99.38 0.0206 2.34 0.141 768 512 393,216 0.0257 23.4 4.69 0.00257 1.170.070 1536 1024 1,572,864 0.00322 11.7 2.34 0.000322 0.586

Mixing or dilution ratios can be established programmably by controllingthe number and distribution of constituent droplets delivered to eachwell. Furthermore, liquid which has been mixed within a well may besubsequently dispensed from that well in the form of unit-sized dropletsfor transport to another well, for example, to perform serial dilutionassays.

8.4.2 Fluid Input

The invention includes a fluidic input module for loading and storage ofsamples and reagents. In one embodiment, a basic input module allowssamples to be loaded using a pipettor or other device and automaticallysubdivides and distributes input fluid as discrete droplets to themultiwell array. When present, the input module serves as the interfacebetween conventional fluid handling technology and the microfluidic chiparchitecture.

The fluid input module generally can include one or more sample loadingreservoirs integrated with the multiwell array. The loading reservoirsmay include a liquid reservoir connected to the network. The loadingreservoirs are interfaced to the outside of the chip and will typicallyhave much larger capacities than individual processing wells where theliquid is completely loaded or they may have smaller capacities butserve as an interconnection between the chip and the outside world wherethe liquid is continually fed. As a general rule, the target capacity ofthe loading reservoirs can be a multiple of the number of wells timesthe unit volume in cases where the liquid is completely loaded.

Thus, at least one unit droplet of each sample or reagent can bedistributed to each well on the plate. Frequently used reagents such asdilution buffers can be loaded in multiple ports for greater parallelismor continually fed in through at least one dedicated loading port.

A general discussion of input reservoirs used in connection with thedroplet microactuator of the present invention can be found hereinbelowwith reference to Section 8.8.6.1.

8.4.3 Screening Capabilities

The invention provides a high-throughput interface for screening oflarge diverse libraries. Clearly, if thousands of compounds must beloaded at the chip's fluidic interface using conventional robotics, thenthe advantage of this platform is severely diminished. As such, while insome embodiments this limited approach was used for loading of smallnumbers of reagents for on-chip titration, in other embodiments a meanswas employed for rapidly populating the wells with a large number ofdifferent compounds. Various alternative embodiments may, for example,include 1) an interface in which droplets of compound are separated byplugs of oil in a long pre-loaded capillary (e.g., glass capillary)which when connected to the chip allows droplets of compound to becaptured and routed on the chip as they are pumped out of the capillary,2) an approach in which compounds are pre-stamped onto the chip andallowed to dry using a high-speed reagent stamping or printing process,or 3) a direct plate to chip interface in which the contents ofconventional 1536 or 384 or 96 well plates can be transported to thechip in parallel by pressurizing the liquid to pass through openings atthe bottom of the wells which are aligned to an array of chip inputs.

In some embodiments, the invention provides electronics and detectorsintegrated directly on the chip substrate to enable higher throughputand rapid readout of results. The use of glass or silicon substratespermit integration of microelectronic circuits and the array of dropletcontrol electrodes on a single substrate. Integration of the driveelectronics onto the chip would vastly increase the available number ofelectronic control signals permitting greater throughput and protocoloptimization. The LCD screen found on a typical PDA such as a PalmPilot™is roughly the same size as a multi-well plate and contains a 480×320array of 153,600 independently controllable electrodes demonstratingthat chip-level integration of electronics can be both feasible andrelatively inexpensive. Integration of electronics onto the substrateprovides the additional advantage that optical or electrochemicalsensors could be integrated with the chip as well.

8.5 Droplet-Based Multiwell Plate Examples

The following non-limiting examples are provided only for the purpose ofillustrating various aspects of this aspect of the invention and shouldnot be construed as limiting the scope of the invention.

8.5.1 Scalable Chip Architecture for Droplet Distribution

8.5.1.1 Well Design

Individual wells can be defined on the chip using the samephotopatternable polymer material (e.g., dry film soldermask) thatdefines the input reservoirs and provides the stand-off between the twoplates in the fully-assembled device. Each well can be designed with atleast a single major opening to permit communication between theinterior of the well and the distribution network for dispensing ofdroplets into and out of the well. Several minor openings may also beincluded to allow air bubbles to escape during filling of the devicewith oil.

The following parameters can be varied to optimize the performance(dispensed droplet volume variation, liquid capacity, ease of fillingwith oil, and resistance to shake-up during handling) of each well:major opening width and length, interior shape and size, location andsize of minor openings, size and shape of electrodes, and chamberheight.

The consistency of dispensed droplet volumes and liquid capacity of thewells have important consequences with respect to the capability ofperforming multi-step dilutions or mixing within the chambers. Forexample, if the volume of the dispensed droplet is found to depend onthe volume of liquid remaining in the well, this issue can be correctedat the program compiler level by accounting for this variation.

8.5.1.2 Transporter Network Design

The droplet distribution network linking the individual chambers isanother component of the multiwell array subsystem of the proposedplatform. While the layout of the network is straightforward owing tothe regularity imposed by the array, electrical connectivity and controlof the electrodes presents a serious optimization challenge. Thus, forexample, in an embodiment in which 10 transport electrodes areassociated with each cell, up to 1,000 independent electrical controlsignals are required. In practice, no more than a few hundred signalscan be inexpensively managed. One solution to this problem is to providemultiplexing of electrode control signals. That is, multiple electrodesare connected to a single signal and activated or deactivated together.For example, a transport line of arbitrary length can be implemented asa line of electrodes where every electrode is connected to one of threesignals which are operated out of phase to transport all droplets on theline in unison. In this manner, many droplets can be arbitrarilymanipulated in parallel, but the design and operation of the device isconsiderably more complex owing to lack of independent control of eachelectrode.

The fabricated multiwell platform can be arranged to provide optimaldroplet routing. Due to the limited number of electronic controlsignals, droplets are preferably not routed independently of each other.In addition, fluidic constraints and overlapping droplet routes willdictate careful droplet scheduling. Computationally tractable algorithmscan be used for the simultaneous coordination of a potentially largenumber of droplets. Limited row-column addressing schemes, where onlyentire rows and columns of droplets can be addressed, can be employed.The insertion of “wait cycles” at appropriate points during sampledelivery, mixing, and dilution can also be employed.

8.5.2 Fluidic Input Module

The fluidic input module can include a smaller array of relatively large(˜10 μL) reservoirs which can be pipette-loaded through access holes inthe chip. The reservoirs store sample or reagent and dispense unit-sizeddroplets on demand for transport to particular well locations. Thedesign of the input module is made challenging both by the need tophysically interface with the outside world and by the need to preciselydispense the unit-sized droplets from a large, arbitrary volume.

Dispensing of unit droplets from the fluidic input is considerably morechallenging than dispensing from the wells because the volume of theliquid reservoir compared to the dispensed droplet is considerablylarger and more variable. Dispensing of droplets becomes inherently morechallenging at larger ratios due to the larger difference in curvaturebetween the initial and final droplets. As described by theYoung-Laplace equation, this curvature difference creates a pressuregradient inside the liquid which opposes further deformation of thesurface. Thus more energy is required to dispense droplets at largeratios (e.g., 100:1) than at smaller ones (e.g., 10:1). Furthermore,larger droplets of liquid are more susceptible to gravitational, buoyantand inertial effects because surface-tension is less dominant at thisscale, leading to less reliable and repeatable manipulation of dropletsby surface-tension-based effects.

In one embodiment, this problem is addressed using a multi-stage inputcomprised of a larger outer reservoir directly connected to a loadinghole and outside world and a smaller inner reservoir connecting theouter reservoir with the multi-well array. The smaller inner reservoiris periodically reloaded from the larger reservoir and dispensesdroplets directly onto the transport network. Dispensing of liquid fromthe larger to smaller reservoir need not be very precise, while therelative stability of the smaller reservoir volume ensuresreproducibility of the unit droplet volume.

For example, one embodiment includes a fluid input module with fixedouter reservoir volume of 10 μL (100 unit droplets of 100 nL each) andinner reservoirs of various volumes ranging from 5 μL (50 unit dropletsof 100 nL each) to 1.0 μL (10 unit droplets of 100 nL each). The entire10 μL volume can be dispensed, reloading the smaller reservoir as manytimes as required, and the volume of each unit-droplet can be measured.The performance of each design in terms of volume reproducibility andthroughput (including reload cycle time) can then be determined.

8.6 Droplet-Microactuator Stamping Platform Overview

One further aspect of the present invention provides a dropletmicroactuator stamping platform. The platform provides, among otherthings, a system for droplet microactuator manipulation (e.g.,dispensing, transport, mixing) of all fluids necessary for preparingsamples for stamping operations. In particular, the invention providesmixing of MALDI matrix on-chip with samples and stamping onto a MALDIplate. FIGS. 12 and 13 illustrate two embodiments of dropletmicroactuator designs of the invention suitable for use as stampingplatforms, wherein FIG. 12 is an electrowetting chip similar to thatused in experiments described herein and FIG. 13 is an electrowettingchip on a 384 well footprint with 16 wells.

The invention includes openings for transporting droplets out of thechip to be stamped on a substrate. For example in one embodiment, theopenings transport the droplets onto a plate. Typically, the surface ofthe plate will be substantially hydrophobic and will include hydrophilicspots where the samples will be deposited, e.g., for MALDI analysissamples will be deposited on these spots and will be interrogated bylaser.

One key aspect of the invention is the ability to transport on a dropletmicroactuator the reagents and samples encountered in a typical MALDI-MSexperiment. It was surprisingly found that droplets including proteinsat concentrations relevant to MALDI-MS can be dispensed from on-chipreservoirs and transported on the droplet microactuator. Further, it wasdiscovered that MALDI matrix containing acetonitrile can be dispensed,transported and manipulated on the droplet microactuator. Moreover, itwas discovered that filler fluids used in electrowetting are compatiblewith MALDI-MS.

Certain embodiments of the droplet microactuator of the invention arecapable of dispensing and transporting sample droplets that includeproteins. A technique was surprisingly discovered which permits dropletswith high concentrations of protein to be manipulated. In oneembodiment, the protein solution has a concentration which exceeds about1 mg/mL, about 10 mg/mL, about 50 mg/mL, or about 100 mg/mL.

Various embodiments of the droplet microactuator are also capable ofmanipulating reagent droplets comprising MALDI matrix. In a preferredembodiment, the droplet microactuator can dispense and transport reagentdroplets comprising acetonitrile. The acetonitrile is typically providedin an amount which is sufficient to keep miscibility of the acetonitrilein the filler fluid at a level which is not unduly detrimental to theeffectiveness of the stamping device. In one embodiment, the MALDImatrix comprises from about 40% to about 75% acetonitrile. In anotherembodiment, MALDI matrix comprises from about 50% to about 70%acetonitrile. In certain embodiments, the MALDI matrix may also includeTFA. For example, in one embodiment, the MALDI matrix includes fromabout 0.05% to about 1% TFA. Thus, for example, in a preferredembodiment, the MALDI matrix includes from about 50% to about 70%acetonitrile and from about 0.05% to about 1% TFA.

The droplet microactuator typically includes a filler fluid, discussedin more detail hereinbelow with reference to Section 8.8.4. The fillerfluid surrounds the droplets being transported. Low viscosity oils arepreferred, preferably silicone oil. The MALDI-MS of silicone oil wasevaluated and it was surprisingly found that the oil is highlycompatible with MALDI-MS, producing no discernable peaks. FIG. 14illustrates the MALDI-TOF MS spectrum of oil used in examples definedherein.

The system of the present invention is generally programmed andconfigured to mix sample droplets with MALDI matrix droplets on-chip toyield MALDI-ready droplets and to direct the MALDI-ready droplets to aport where some portion or the entire droplet can exit the dropletmicroactuator to be deposited on a MALDI substrate. In operation,droplets of sample and MALDI matrix are dispensed from on-chipreservoirs, mixed and spotted onto a substrate through an opening in thetop plate (see aperture 1518 in FIG. 15). Passive stamping sometimesresults in dead volume in the opening. In one embodiment, this problemis resolved by adding a small quantity of oil through one of the oilloading ports which provides enough pressure to force the liquid out ofthe opening completely.

In one embodiment, the sample and the MALDI matrix are merged at astamping hole. Mixing is significantly faster using this approach due tothe larger surface area available for diffusion in the hole.

Preferably, cross-contamination is reduced to a level at which it doesnot significantly impact analysis of deposited samples. In someembodiments, cross-contamination is reduced by reducing the acetonitrileconcentration in the sample by dilution. Cross-contamination may also bereduced by minimizing the overlapping paths between different samples.

8.6.1 Droplet Manipulation

The stamping platform of the invention makes use of a dropletmicroactuator chip which is useful for effecting droplet operations,such as dispensing, splitting, transporting, merging, mixing, agitating,and the like. Preferably, droplet manipulation is accomplished usingelectric field mediated actuation, as described hereinbelow withreference to Section 8.8. As illustrated in FIG. 15, the basic dropletmicroactuator 1500 includes two parallel plates, top plate 1502 andbottom plate 1504, separated by a gap 1506 formed such as through theuse of spacers 1508. One or both of the plates 1502, 1504 includes oneor more paths or networks of substantially planar control electrodes1512 and corresponding reference electrodes 1514 for performing dropletmanipulations. Droplets 1516 are interposed in the space 1506 betweenthe plates 1502, 1504 on these paths or networks. Space 1506 surroundingthe droplets is typically filled with a filler fluid as describedhereinabove. Top plate 1502 may include a stamping aperture 1518 for thepassing of a droplet 1516 through to a stamping plate 1522. The dropletmicroactuator 1500 works with a wide variety of liquid droplets, thoughconductive liquids are preferred.

Droplet transport for stamping occurs along the path or network ofcontrol electrodes. The path or network requires interconnections toelectrically connect electrodes to contact pads for connection toexternal circuitry and may also include interconnections for connectingcertain electrodes together. The droplet microactuator operates bydirect manipulation of discrete droplets, e.g., using electrical fields.By applying different voltages to adjacent control electrodes, a localand electrically controllable energy gradient can be established. Adroplet adjacent to an energized electrode will move to align itselfwith the energized electrode, i.e., the droplet will be transported tothe position of that electrode. A series of successive transfers willtransport droplets along the path or network of control electrodes. Inaddition to transport, other operations including merging, splitting,mixing and dispensing of droplets can be accomplished in the same mannerby varying the patterns of voltage activation.

Further description of architecture and operations involving a dropletmicroactuator of the present invention can be found hereinbelow withreference to Section 8.8.

8.7 Droplet-Microactuator Stamping Platform Examples The ensuingexamples are illustrative of various embodiments of the invention, andshould not be interpreted as limiting the scope of the invention. Theexamples provide, among other things, a demonstration of the feasibilityof the droplet microactuator stamping platform described herein,including validation of on-chip manipulation (dispensing, transport,mixing) of all fluids of interest for the proposed application on acoplanar platform, and mixing of MALDI matrix on-chip with samples andstamping onto a MALDI plate.

8.7.1 Material and Methods

Protein samples were provided by LEAP or were obtained from SIGMA.MALDI-TOF MS was done by Global Peptide Services, Colorado. FIG. 12shows a chip used in the experiments. FIG. 16 shows time lapsed imagesof a stamping experiment accomplished using the chip of FIG. 12. FIG. 13shows another chip which was fabricated on a 384-well pitch with 16wells.

8.7.2 Material Compatibility Studies

The compatibility of the various materials encountered in a typicalMALDI-MS experiment was established by demonstrating the following.

1) Dispensing and transport of a representative set of protein dropletsat concentrations relevant to MALDI-MS from on-chip reservoirs.

2) Dispensing and transport of MALDI matrix which contains acetonitrile.

3) Compatibility of filler fluids used in electrowetting with MALDI-MS

8.7.2.1 Protein Compatibility

Table 11 lists proteins that were tested on the system along with thehighest tested concentrations. Note that some of these concentrationsare the highest “tested” and up to 10 s of mgs/mL of protein has beensuccessfully dispensed and transported on the chips. This is threeorders of magnitude higher than what is typically seen in MALDI-MSapplications. TABLE 11 Protein compatibility chart Protein Highestconcentration tested BSA 90 fmol/uL (6 ug/mL) Bovine insulin 100 pmol/uL(573 ug/mL) Lysozyme 75 mg/mL Glucose isomerase 10 mg/mL Proteinase K 10mg/mL

The BSA sample was in 25% ACN (provided by LEAP Tech) and the bovineinsulin (from SIGMA) was in 1% TFA. In addition to the above proteins,the manipulation of sample matrices containing high concentration ofproteins such as whole blood, plasma and serum have been shown.

8.7.2.2 Solvent/MALDI Matrix Compatibility

The most commonly used solvent to prepare a MALDI matrix is 50-70%acetonitrile with 0.05-1% TFA. Though 100% acetonitrile is partiallymiscible (3-5%) in the oils typically used for electrowetting, a mixtureof acetonitrile and water (up to 70% ACN) was not measurably miscible inoil. Droplets were successfully manipulated containing up to 70%acetonitrile on the system. MALDI matrix droplets containing 50% ACN and0.05% TFA was also successfully dispensed and manipulated on the chip.

8.7.2.3 Filler Fluid Compatibility

In order to prevent fouling of the surfaces, the use of low viscosityoils is preferred as the filler media surrounding the droplets. TheMALDI-MS of the silicone oil commonly used was evaluated and nodiscernable peaks >3000 Da were found. FIG. 14 shows the mass spectra ofoil. The small peak which is visible at 8212 Da is from the MALDImatrix.

8.7.3 System scaling and integration

System integration and scaling was demonstrated as follows:

1) Mixing of protein droplets with MALDI matrix droplets on-chip.

2) Passive stamping through an opening or an “open” system onto a topplate which is hydrophobic all over the surface except for thehydrophilic spots where the MALDI samples will be interrogated by laser.

3) Evaluating the effect of cross contamination between the dropletseither mediated through oil or the surfaces.

4) Determining the effect of aspect ratios on stamping.

5) Investigating use of droplets digitized and deposited directly fromthe well plate obviating the need for a top plate. Determine operationallimitations of this approach.

8.7.3.1 Stamping of Single Sample

FIG. 15 illustrates the setup used to perform the MALDI stampingexperiment. BSA (90 nM in 25% ACN) was used as the model protein for thestamping experiments. Droplets of sample and MALDI matrix were formedfrom on-chip reservoirs, mixed and passively spotted onto a glass platethrough an opening in the top plate. The droplets were approximately 100nL each. Passive stamping sometimes resulted in dead volume being leftover in the opening. This problem was resolved by adding a smallquantity of oil through one of the oil loading ports which providesenough pressure to force the liquid out of the hole completely.

In a variation of the stamping experiment described above, the sampleand the MALDI matrix were merged at the stamping hole. Mixing isexpected to be significantly faster using this approach due to thelarger surface areas available for diffusion in the hole.

8.7.3.2 Stamping of Multiple Samples

The previous experiment was scaled up to demonstrate the passivestamping of four different BSA droplets. The mixing with the matrix wasdone at the stamping hole in this experiment.

8.7.3.3 Cross-Contamination Studies

Cross-contamination studies were performed to evaluate the carryover ofprotein samples from one droplet to another. In the first experiment,several droplets (total volume ˜500 nL) of 100 pmol/uL insulin (totalinsulin manipulated—50 pmol) in 1% TFA were moved across 6 electrodesseveral times and collected through the stamping hole. The total timethe insulin droplets resided on the 6 electrodes was approximately 5minutes. A water droplet was also moved on the same path as the proteindroplet for approximately 5 minutes and collected through a differentstamping hole. 5 uL of matrix was added to both the samples andMALDI-TOF MS was done by spotting the entire sample. FIG. 17 illustratesMALDI-MS for a blank droplet after cross-contamination experimentswithout acetonitrile in the system. The figure shows the spectrum and nopeaks are visible at the molecular weight of insulin. The lowestdetectable concentration obtained by doing MS analysis on a dilutioncurve was 2.5 fmol of insulin. As such, the cross-contamination from a50 pmol sample is below 2.5 fmoles (carryover of 0.005%) and is notdetectable by the instrument used.

In the second experiment the protein sample and a blank sample were bothpremixed with the MALDI matrix to see if the presence of acetonitrileincreased the possibility of contamination. Four 100 nL droplets of 100pmol/uL insulin (40 pmol) with matrix were transported across 12electrodes serially and discarded to waste. Four 100 nL droplets of theblank droplet (with matrix) was moved along the same path and collectedthrough a stamping hole. The collected droplet was stamped as is withoutany additional matrix added to it. FIG. 18 illustrates MALDI-MS for ablank droplet after cross-contamination experiments without acetonitrilein the system according to this experiment. The figure shows the zoomedmass spectrum of the blank droplets and a peak is visible at themolecular weight of insulin. This indicates that there is somecross-contamination. To estimate the levels of cross-contamination a logdilution series of insulin between 2.5 fmoles and 250 pmoles was alsoanalyzed using MALDI-MS. The intensity of the peak (baseline corrected)for the different samples analyzed is shown in Table 12. TABLE 12Mass-spec peak intensity for different samples Peak intensity (baselineSample corrected) 250 pmol 1743  25 pmol 4339  2.5 pmol 5816 250 fmol333  25 fmol 114  2.5 fmol no peak Blank 383

From the table, it can be inferred that the cross-contamination from a40 pmol sample is around 250 fmol which amounts carryover of 0.625%. Asimilar carryover was seen when both the sample and the blank werecollected through the same hole.

This cross-contamination can be attributed to the presence ofacetonitrile in the samples (from the matrix) since no contamination wasobserved using an aqueous sample of proteins. Without wishing to bebound by a particular theory, it is suspected that microdroplets areejected during manipulation. It is believed that by changing thegeometry of the electrodes, the ejection of microdroplets can be reducedor eliminated. Cross-contamination can also be minimized by reducing theacetonitrile concentration in the sample by dilution, and by minimizingthe overlapping paths between different samples.

8.7.4 Stamping Platform Conclusions

Among other things, the work described herein demonstrates thefeasibility of the stamping platform of the invention. A dropletmicroactuator of the invention can dispense and transport proteinsamples with high protein content, MALDI-specific solvents, and MALDImatrix. An oil matrix can be used to prevent proteins from sticking toelectrodes. There are limitations on the percentage of acetonitrilesolvent in the sample liquids used but these limits do not substantiallyimpair the utility of the device. Mixing of the MALDI matrix with samplematerials on chip can be accomplished as expected. Stamping through ahole in the top plate is a feasible embodiment of the invention. Lessthan 1% carryover appears to occur when very low protein concentrationsolution droplets follow the same path as high protein concentrationsolution droplets.

8.8 Droplet Microactuator Architecture and Operation

The various aspects of the present invention discussed hereinabovegenerally include a droplet microactuator controlled by a processor. Forexample, the processor may, among other things, be programmed to controldroplet manipulations on a droplet microactuator. A wide variety ofdroplet microactuator configurations is possible. Examples of componentswhich may be configured into a droplet microactuator of the inventioninclude various filler fluids which may be loaded on the dropletmicroactuator; fluid loading mechanisms for introducing filler fluid,sample and/or reagents onto the droplet microactuator; variousreservoirs, such as input reservoirs and/or processing reservoirs;droplet dispensing mechanisms; means for controlling temperature of thedroplet microactuator, filler fluid, and/or a droplet on a dropletmicroactuator; and magnetic field generating components for manipulatingmagnetically responsive beads on a droplet microactuator. This sectiondiscusses these and other aspects of the droplet microactuator and theiruse in the systems of the invention.

8.8.1 Droplet Microactuator

The various aspects discussed hereinabove can make use of a dropletmicroactuator, sometimes referred to herein as a chip. The dropletmicroactuator can include a substrate with one or more electrodesarranged for conducting one or more droplet operations. In someembodiments, the droplet microactuator can include one or more arrays,paths or networks of such electrodes. A variety of electrical propertiesmay be employed to effect droplet operations. Examples includeelectrowetting and electrophoresis.

In one embodiment, the droplet microactuator includes two or moreelectrodes associated with a substrate, and includes a means forpermitting activation/deactivation of the electrodes. For example, theelectrodes may be electronically coupled to and controlled by a set ofmanual switches and/or a controller. The droplet microactuator is thuscapable of effecting droplet operations, such as dispensing, splitting,transporting, merging, mixing, agitating, and the like. Dropletmanipulation is, in one embodiment, accomplished using electric fieldmediated actuation. Electrodes will be electronically coupled to a meansfor controlling electrical connections to the droplet microactuator.

The basic droplet microactuator includes a substrate including a path orarray of electrodes. In some embodiments, the droplet microactuatorincludes two parallel substrates separated by a gap and an array ofelectrodes on one or both substrates. One or both of the substrates maybe a plate. One or both substrates may be fabricated using PCB, glass,and or semiconductor materials as the substrate. Where the substrate isPCB, the following materials are examples of suitable materials: MitsuiBN-300; Arlon 11N; Nelco N4000-6 and N5000-30/32; Isola FR406,especially IS620; fluoropolymer family (suitable for fluorescencedetection since it has low background fluorescence); and the polyimidefamily. Various materials are also suitable for use as the dielectriccomponent of the substrate. Examples include: vapor depositeddielectric, such as parylene C (especially on Glass), and parylene N;Teflon AF; Cytop; and soldermasks, such as liquid photoimageablesoldermasks (e.g., on PCB) like Taiyo PSR4000 series, Taiyo PSR AUSseries (good thermal characteristics for applications involving thermalcontrol), and Probimer 8165 (good thermal characteristics forapplications involving thermal control); dry film soldermask, such asthose in the Dupont Vacrel family; and film dielectrics, such aspolyimide film (Kapton), polyethylene, and fluoropolymers like FEP,PTFE. Some or all of the substrate may also include a hydrophobiccoating. Suitable examples include Teflon AF; Cytop; coatings in theFluoropel family; silane coatings; fluorosilane coatings; and 3M Novecelectronic coatings.

Where the droplet microactuator includes two plates, droplets may beinterposed in the space between the plates. Space surrounding thedroplets typically includes a filler fluid. The droplet microactuatorcan conduct droplet operations using a wide variety of fluid droplets,though conductive fluids are preferred. Filler fluids are discussed inmore detail hereinbelow with reference to Section 8.8.4.

Surfaces of the droplet microactuator are typically coated with ahydrophobic coating. For applications involving thermal cycling, ahydrophobic coating should be selected that is resistant to thermalstress during prolonged thermocycling operation. Examples of suitablethermal resistant materials include soldermasks such as Probimer® 8165which has been developed for use in the automotive industry and hasexcellent thermal shock resistance, and PCB board materials such asMitsui BN-300 which is resistant to high temperature and warpage.

Droplet transport occurs along a path or network of control electrodes.The array or path includes electrical connections for electricallycoupling electrodes to external circuitry. The array or path may alsoinclude electrical connections for electrically coupling certainelectrodes together. The electrodes can be controlled via the externalcircuitry by a processor. Droplet operations may be effected bysupplying voltage to the electrodes. While the preferred voltage variesdepending on the thickness of the dielectric, for a dielectric constantin the range of 2-100 and thickness in the range of 1 nm to 10 mm, thepreferred energy per unit area limits are in the range of about 300microjoule/sq meter to about 300000 microjoule/sq meter. The preferredactivation voltage is in the range of about 1 mV to about 50 kV, orabout 1V to about 10 kV, or about 5V to about 1000V, or about 10V toabout 300V.

Typically, the electrodes are fired via a voltage relay. The dropletmicroactuator operates by direct manipulation of discrete droplets,e.g., using electrical fields. For example, a droplet adjacent to anenergized electrode with surrounding electrodes grounded will transportto align itself with the energized electrode, i.e., the droplet will betransported to the position of that electrode. A series of successivetransfers will transport droplets along the path or network of controlelectrodes. In addition to transport, other operations includingmerging, splitting, mixing and dispensing of droplets can beaccomplished in the same manner by varying the patterns of voltageactivation.

It should be noted that electrodes can be activated in a variety ofways. For example, an electrode can be activated by applying a DCpotential. Similarly, an electrode can be activated by applying an ACpotential, so that the activated electrode has an AC potential and anunactivated electrode has a ground or other reference potential. Inanother aspect, the potential may be applied by repeatedly activating anelectrode and then inverting it. An AC mode can be effected by usingsoftware to rapidly switch between polarities of the outputs.

In some embodiments the invention employs droplet operation structuresand techniques described in U.S. Pat. No. 6,911,132, entitled “Apparatusfor Manipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on Jan. 30, 2006; U.S. Pat. Nos.6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and 6,565,727, entitled“Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24,2000, both to Shenderov et al.; U.S. Patent Publication No. 20060254933,entitled “Device for transporting liquid and system for analyzing”published on Nov. 16, 2006 to Adachi et al.; International PatentApplication No. PCT/US 06/47486, entitled “Droplet-Based Biochemistry,”filed on Dec. 11, 2006; and International Patent Application No. PCT/US06/47481, entitled “Droplet-Based Pyrosequencing,” filed on Dec. 11,2006, the disclosures of which are incorporated herein by reference fortheir teachings concerning structures and techniques for conductingdroplet operations.

Droplet operations can be rapid, typically involving average linearvelocities ranging from about 0.01 cm/s to about 100 cm/s, or from about0.1 cm/s to about 10 cm/s, more preferably from about 0.5 cm/s to about1.5 cm/s. Moreover, droplets may typically be manipulated at a frequencyof manipulation ranging from about 1 Hz to about 100 KHz, preferablyfrom about 10 Hz to about 10 KHz, more preferably from about 25 Hz toabout 100 Hz. In addition to being rapid, droplet manipulations usingthe droplet microactuator are also highly precise, and multiple dropletscan be independently and simultaneously manipulated on a single dropletmicroactuator.

Discrete droplet operations obviate the necessity for continuous-flowarchitecture and all the various disadvantages that accompany such anarchitecture. For example, near 100% utilization of sample and reagentis possible, since no fluid is wasted in priming channels or fillingreservoirs. Further, as noted above, droplet movement can be extremelyrapid. The droplet microactuator may in some cases be supplemented bycontinuous flow components and such combination approaches involvingdiscrete droplet operations and continuous flow elements are within thescope of the invention. Continuous flow components may be controlled bythe controller. Nevertheless, in certain other embodiments, variouscontinuous flow elements are specifically avoided in the dropletmicroactuator of the invention and/or methods of the invention. Forexample, in certain embodiments, one or more of the following componentsis excluded from a droplet microactuator and/or methods of theinvention: microchannels; fixed microchannels; networks ofmicrochannels; pumps; external pumps; valves; high-voltage supplies;centrifugal force elements; moving parts.

Electric field mediated actuation also obviates the need for otherdroplet operations and all the various disadvantages that accompany suchtechniques. It will be appreciated that the droplet microactuator maynevertheless be complemented or supplemented with other dropletmanipulation techniques, such as electrical (e.g., electrostaticactuation, dielectrophoresis), magnetic, thermal (e.g., thermalMarangoni effects, thermocapillary), mechanical (e.g., surface acousticwaves, micropumping, peristaltic), optical (e.g., opto-electrowetting,optical tweezers), and chemical means (e.g., chemical gradients). Whenthese techniques are employed, associated hardware may also beelectronically coupled to and controlled by the controller. However, inother embodiments, one or more of these droplet operation techniques isspecifically excluded from a droplet microactuator of the invention.

The droplet microactuator can be manufactured in a highly compact formand can be driven using a very small apparatus. For example, dropletmicroactuator and apparatus may together be as small as several cubicinches in size. The droplet microactuator requires only small amounts ofelectrical power and can, for example, readily be operated usingbatteries. The droplet microactuator can perform droplet operationsusing extremely small droplets. Droplets are typically in the range offrom about 1 fL to about 1 mL, more preferably from about 100 μL toabout 1 μL, still more preferably from about 10 nL to about 1 μL.

The use of discrete droplets for on-chip processing instead ofcontinuous flows provides several important advantages. Since samplefluid need not be expended for priming of channels or pumps virtuallyall of the sample fluid can be used for analysis and very small volumesof sample (e.g., less than about 100 μL or less than about 50 μL or lessthan about 25 μL) can be analyzed. The same advantages apply to the useof reagents where reducing the volume of reagents consumed has theadvantage of reducing the cost of the analysis. The use of discretesmall-volume droplets also permits a large number of reactions toperformed in a small footprint (e.g. greater than 10 per cm² or greaterthan 100 per cm or greater 1,000 per cm² or greater than 10,000 percm²).

Various components of the invention may be included as components of thedroplet microactuator. In fact, an entire system of the invention may beprovided as an integrated droplet microactuator. In some embodiments,the droplet microactuator includes various sensors and means forelectronically coupling the sensors to external circuitry. In otherembodiments, the droplet microactuator includes heaters and/or magneticfield generating elements and means for coupling such elements toexternal circuitry. Further, a droplet microactuator including any oneor more of the reagents described herein in a reservoir or in dropletform is also an aspect of the invention.

Optical windows can be patterned in the electrodes to enhance thecapability of performing optical detection on the chip. Where theelectrode is formed in an opaque material on a transparent substrate, awindow in the electrode can be created permit light to pass through thesubstrate. Alternatively, when the electrode material is transparent, amask can be created to eliminate stray light. Additionally, the openingcan be patterned as a diffraction grating. Adaptive optical windows canbe created as well, using a second electrowetting layer. For example,opaque oil (e.g. oil dyed black) can be used with a transparent dropletto create a temporary and movable optical window.

8.8.2 Droplet Microactuator Fabrication

Droplet microactuators can be made using standard microfabricationtechniques commonly used to create conductive interconnect structures onmicrodroplet microactuators and/or using printed-circuit board (PCB)manufacturing technology. Suitable PCB techniques include thosedescribed in U.S. patent application Ser. No. 11/343,284, entitled“Apparatuses and Methods for Manipulating Droplets on a Printed CircuitBoard,” filed on Jan. 30, 2006, the entire disclosure of which isincorporated herein by reference. These techniques permit the dropletmicroactuator to be manufactured in bulk at very low cost. Low costmanufacture enables economical production of droplet microactuators,even for use as one-use disposables. Thus, the invention provides amethod in which droplet microactuators are supplied to users ascomponents of disposable cartridges for use in systems of the invention.

Designs can also be implemented on glass or silicon using conventionalmicrolithography techniques with the capability of producing muchsmaller features than are typical in a PCB process. Even, for example,for a 1,572,864-reservoir droplet microactuator with 70 μm reservoirspacing and 3 fL reservoir volume, the minimum required lithographicfeature size is ˜0.5 μm which is well within the capabilities ofconventional microlithographic techniques currently used in thesemiconductor industry.

Because the chip can be loaded directly using manual or robotic pipettedispensers and can be analyzed using standard plate reading equipment,it will easily integrate into existing laboratory work flows. This is asignificant advantage over other microfluidic approaches which mayrequire adaptation of the assays to continuous-flow format orspecialized equipment for sample handling and read-out.

8.8.3 Cartridge

In some embodiments, the invention includes a cartridge for coupling tothe droplet microactuator. It will be appreciated that a cartridge,while not necessary to the operation of the invention, may be convenientin some circumstances. When present, the cartridge may include a meansfor electrically coupling the path or network of the dropletmicroactuator to a processor, e.g., a processor of a dropletmicroactuator system of the invention. In this embodiment, theelectrical connection is: electrodes—cartridge—processor, where theremay be additional elements between the three. In another embodiment, thecartridge may include means for physically coupling to the dropletmicroactuator. In this embodiment, the electrical connection may be:electrodes—processor—cartridge. Alternatively, the cartridge may lackelectrical components altogether.

When present, the cartridge may include reservoirs for one or morereagents, e.g., pre-loaded reagents. The droplet microactuator may beconfigured so that a fluid path may be established between the cartridgereservoirs and the interior of the droplet microactuator for flowingreagents, sample and/or filler fluid from the cartridge onto the dropletmicroactuator. For example, preloaded cartridge reservoirs may bedispensed into the droplet microactuator prior to, during, or aftercoupling of the cartridge to the analyzer. The cartridge may be sealed,self-contained and/or disposable. It may be supplied with or without adroplet microactuator. Such cartridges can be used to ensure repeatableassay conditions, permit safe handling and disposal of infectious orhazardous material, and/or reduce cross-contamination between runs. Thecartridge may, for example, include a machined plastic part. It may beaffixed to and provided in combination with the droplet microactuator.

The cartridge materials are selected to provide storage of reagentswithout degradation or contamination of the reagents. Moreover, theyshould be selected to provide reliable operation at elevated temperatureand to ensure compatibility with the real-time chemistry. They may, forexample, include molded plastic components. In some embodiments, sealed,disposable test cartridges enhance operator safety and facilitate safedisposal.

Various components of the droplet microactuator system may be includedon the cartridge. For example, the top-plate, which encloses theinterior space of the droplet microactuator, may be provided as acomponent of the cartridge. Various sensors may also be included ascomponents of the cartridge.

8.8.4 Filler Fluid

The droplet microactuator of the invention includes one or more free(i.e., fluid-fluid) interfaces. Examples include a liquid-liquid orliquid-gas interface. Typically chemistry is performed in the primary(droplet) phase, and the secondary phase serves as a filler fluidseparating the droplets from each other. The secondary phase can, forexample, be a liquid, gel, and/or a gas. Where the secondary phaseincludes a liquid, the liquid is sufficiently immiscible with theprimary liquid phase to permit the droplet microactuator to conduct oneof more droplet operations.

It should also be noted that the droplet microactuator may include morethan two phases. For example, in one embodiment the dropletmicroactuator operates based on an aqueous-oil-air three-phase system.In a related environment, the droplet microactuator may operate based onan aqueous-first oil-second oil three-phase system, such as a systemincluding an aqueous droplet surrounded by silicon oil, which is in turnsurrounded by a fluorosilicon oil. Generally, three-phase systems willinclude three components which are mutually immiscible or substantiallyimmiscible.

In another embodiment, oil or another immiscible liquid may be used as adroplet encapsulant for electrowetting. For example, a droplet can beencapsulated in a shell of oil by moving the droplet through an air/oilinterface. Each droplet would then have its own local bath of oil withthe space between encapsulated droplets filled with either air or athird immiscible liquid. Among other advantages, this approach is usefulfor minimizing the transfer of material between droplets in the systemby partitioning into the oil phase while retaining the advantageousproperties of the oil with respect to evaporation and fouling of thesurface. This approach may also be used to facilitate electrowetting ofnon-electrowettable liquids which are immiscible with electrowettableliquids. In a specific embodiment of this concept the immiscible liquidcan be chosen to be crosslinkable (by UV, heat, moisture or chemically)to create capsules of liquids with solid shells, for drug deliverysynthesis applications.

Further, in some applications it may be desirable or necessary toperform certain operations in an immiscible liquid, such as oil, andothers in air. The invention includes hybrid systems in which dropletmanipulation is performed both in air and in an immiscible liquid fillerfluid such as oil. For example, samples may be processed under oil andthen transported into an air-medium portion for evaporation forsubsequent analysis, for example, by MS. Conversely, a sample could becollected in air and then processed with droplets under oil. Thus, thedroplet microactuator may include a transport path for moving dropletsfrom a droplet microactuator surface in a space filled with filler fluidto a droplet microactuator open to the atmosphere or including a gaseousfiller fluid.

The filler fluid may be any fluid in which the droplet microactuatorcan, under the right conditions, conduct one or more droplet operations.It should be noted that certain filler fluids may be solids or highlyviscous fluids under certain conditions, e.g., during transport, whilethey are transformed into fluids for operation, e.g., by heating. Thefiller fluid may be a liquid or gas during operation of the dropletmicroactuator. Examples of suitable liquid filler fluids include,without limitation, silicone oils; fluorosilicone oils; hydrocarbons,including for example, alkanes, such as decane, undecane, dodecane,tridecane, tetradecane, pentadecane, hexadecane; aliphatic and aromaticalkanes such as dodecane, hexadecane, and cyclohexane, hydrocarbon oils,mineral oils, paraffin oils; halogenated oils, such as fluorocarbons andperfluorocarbons (e.g. 3M Fluorinert liquids); mixtures of any of theforegoing oils in the same class; mixtures of any of the foregoing oilsin different classes. Examples of suitable gas filler fluids include,without limitation, air, argon, nitrogen, carbon dioxide, oxygen,humidified air, any inert gases. In one embodiment, the primary phase isan aqueous solution, and the secondary phase is air or an oil which isrelatively immiscible with water. In another embodiment, the fillerfluid includes a gas that fills the space between the plates surroundingthe droplets. A preferred filler fluid is low-viscosity oil, such assilicone oil. Other suitable fluids are described in U.S. patentapplication Ser. No. 11/639,594, entitled “Filler Fluids for DropletOperations” filed on Dec. 15, 2006, the entire disclosure of which isincorporated herein by reference. The fluid may be selected to preventany significant evaporation of the droplets.

The phases of the fluids used in the protocols of the invention may beselected to facilitate protocols of the invention without undueformation of bubbles, loss of reagent to the filler fluid, and/oradherence of reagent to the droplet microactuator surface.

In certain embodiments of the invention the filler fluid may be selectedto reduce or prevent evaporation of sample, reagent, or other dropletsutilized in the protocols of the invention. The filler fluid may beselected to prevent sample, reagent, or other droplets utilized in theprotocols of the invention from evaporating and becoming too small forfurther effective manipulation. Similarly, the filler fluid can beselected to prevent evaporation of sample, reagent, or other dropletsutilized in the protocols of the invention from detrimentallyconcentrating species within the droplets in a manner which results inan unduly adverse affect on the intended use of the droplet. Moreover,the filler fluid may be selected to reduce or prevent transport ofmaterial from sample, reagent, or other droplets utilized in theprotocols of the invention across the phase boundary to maintain dropletvolume and/or ensure reliable microfluidic operation and/or assayresults. Miscibility between phases can sometimes result in shrinking(or swelling) of the droplet phase. To prevent or reduce this problem,one or more phases of the system may be saturated with the equilibriumconcentration of another phase to reduce shrinking or swelling. Thus,for example, the filler fluid may be saturated with the equilibriumconcentration of the solvent for sample, reagent, or other dropletsutilized in the protocols of the invention, and/or one or more of thesample, reagent, and/or other droplets utilized in the protocols of theinvention may be saturated with the equilibrium concentration of thefiller fluid.

In some embodiments, a liquid filler fluid is selected to minimizecontact between the droplet and droplet microactuator surfaces. That is,a film of liquid may exist between the droplet and surface whichprevents material within the droplet from coming into contact with andadhering to the coated surface. This approach helps to prevent foulingof the surface and related interference with droplet transport. Forexample, it has been observed that high concentrations of certainproteins in water droplets readily stick to certain hydrophobic surfacesspoiling the hydrophobic nature of these surfaces; whereas, the samedroplets can be moved across the same surfaces without appreciableadhesion of proteins if bathed in an oil which minimizes contact betweenthe two surfaces. This approach may also help to avoidcross-contamination between droplets caused by deposition of materialfrom one droplet which is then picked up by a second droplet. In asimilar embodiment, a film between the droplet and droplet microactuatorsurface can be used to lubricate the droplet by preventing friction-likephysical interactions between the droplet and surface during dropletoperations.

In one embodiment, the invention provides a thin coating of a liquidfiller fluid layer in an otherwise gas filled system. For example, theinvention provides a microfluidic system including an open or enclosedsystem including a thin layer of filler fluid, such as oil, layered on adroplet microactuator surface, wherein the system is otherwise filledwith a gas. The oil is of sufficient thickness to provide lubricationand contamination of droplet microactuator surfaces and contamination ofdroplets via droplet microactuator surfaces. Preferably the oil isselected to minimize transport of material between the droplet and oilphases. One advantage of this approach is reduction of carry-over in thedroplet microactuator. The surface may in some embodiments be treated bycoating it with the filler fluid while operating in air. This approachis also useful for loading operations as a means to retain thelubricating effect of oil while avoiding trapping of oil bubbles in thebulk filler fluid.

Treatment of a Teflon AF surface with silicone oil can provide some ofthe lubrication benefit of silicone oil filler fluid even when operatingin air. This approach can be used to prime the droplet microactuatorwith a lubricating layer of oil, followed by replacement with air toallow samples to be loaded without introduction of bubbles, followed byre-introduction of oil to prevent evaporation of the samples. Thus thebenefits of each kind of system are available depending on the type ofmicrofluidic processing to be carried out.

In another embodiment, the filler fluid can be completely exchanged atdifferent steps within a protocol. For example, a gas filler fluid canbe introduced during sample loading to prevent trapping of air bubblesand then a liquid filler fluid can be pumped in to prevent evaporationof the liquid. Different types of filler fluid can be pumped into or outof the system depending on the particular assay steps to be performed.

In yet another embodiment, multiple filler fluids can be used within asingle system. For example, a droplet microactuator can be selected tohave separate gas filled and liquid filled regions. Operations orcertain types of droplets can be segregated between the different fillerfluid regions.

The filler fluid may be selected based on its refractive index to eithermatch the droplet to prevent refraction of light passing through or nearthe droplet. Alternatively the filler fluid may be selected with arefractive index that differs from the droplet to provide contrast forcertain types of optical measurements or optical manipulations. A fillerfluid may be chosen to have a lower index of refraction than the primaryliquid so that light can be transmitted though the primary liquid bytotal internal reflection. The primary phase can include highlyelongated droplets which can serve as “light pipes” to convey lightbetween two locations, e.g. to facilitate optical analyses.

The filler fluid may be selected based on its color to facilitate director indirect visualization of the droplet, e.g., by providing contrastbetween the sample, reagent, and/or other droplets used in the protocolsof the invention and the filler fluid. This approach can enhancevisualization of the different phases, for example to distinguishdroplets from filler fluid or from air bubbles. In optical applications,the differential absorbance of the two phases can be used to modulatethe color of light passing through the system. As another example, inapplications where fluorescence measurements are made within droplets itmay desirable for the oil to include molecules, such as dyes, thatabsorb the emitted wavelength of light to minimize cross-talk betweenreactions occurring in adjacent droplets.

The filler fluid may be selected to have particular thermal propertiesthat can either thermally insulate the droplets or conduct heat awayfrom the droplets. For example, in the amplification protocols of theinvention, a thermally conductive or low heat capacity filler fluid maybe desirable to permit rapid changes in temperature. For applicationswhere a steady temperature is required, a thermally insulating or highheat capacity filler fluid can be used to provide temperature stability.

The filler fluid may be selected to undergo a phase change uponpresentation of an appropriate stimulus. For example, a wax-like fillerfluid (e.g. paraffin wax or octadecane) can be used where the fillerfluid is changed from solid to liquid form by application of heat.Lowering the temperature would return the filler fluid to a solid sothat droplets would be contained within a solid matrix. Encapsulation ofthe liquid phase within a solid may facilitate storage and handling ofthe sample, reagent, and/or other droplets utilized in the protocols ofthe invention and/or allow for safe and convenient disposal of thematerials following use of the droplet microactuator. The filler fluidcan be stored as a solid on the droplet microactuator, in acartridge-based reservoir, or elsewhere, and heated to permit the fluidto flow into and fill the droplet microactuator. Or the immisciblefiller fluid can be selected to be crosslinkable (by UV, heat, moistureor chemically) to create capsules of liquids within a solid shell.

The filler fluid may be selected to have particular gas permeability orsaturation properties. In certain applications a reaction occurringinside the droplet may consume oxygen or other gas which may need to bereplenished by gas contained within or transported through the fillerfluid. For example, some fluorinated oils have useful gas permeabilityproperties for such applications. Alternatively, the filler fluid may beselected to exclude certain gases from the droplet, for example tomaintain anaerobic conditions within the droplet. The filler fluid maybe selected to have a certain degree of miscibility or partitioning intothe droplet phase. Usually, complete or substantially complete lack ofmiscibility between the droplet and filler fluid is desired, but someapplications may benefit from some limited degree of miscibility betweenthe phases or partitioning of particular molecules between the phases,e.g., liquid-liquid extraction applications. In certain applicationswhere dissolved gases in the filler fluid may be problematic, a meansfor degassing the filler fluid prior to or during use may need to beprovided. For example, filler fluid may be degassed by incubation undervacuum, heating, sparging or by centrifugation.

The filler fluid may be selected to have a particular surface orinterfacial tension with the droplet phase or with the dropletmicroactuator surfaces. Surfactants can be added to the filler fluid tostabilize liquid films that may be present between the droplet and solidphases. Examples of suitable surfactants include nonionic low HLB(hydrophile-lipophile balanced) surfactant. The HLB is preferably lessthan about 10 or less than about 5. Suitable examples include: TritonX-15 (HLB=4.9); Span 85 (HLB 1.8); Span 65 (2.1); Span 83 (3.7); Span 80(4.3); Span 60 (4.7); and fluorinated surfactants.

Surfactants are preferably selected and provided in an amount which (1)results in more droplet operations on the droplet microactuator ascompared to corresponding droplet microactuator without the surfactant;or (2) makes one or more droplet operations possible on the dropletmicroactuator as compared to corresponding droplet microactuator withoutthe surfactant; or (3) makes one or more droplet operations morereliable on the droplet microactuator as compared to correspondingdroplet microactuator without the surfactant. In a related example,surfactants are preferably selected and provided in an amount whichmakes one or more droplet operations possible or more reliable fordroplets including one or more specific reagents or mixtures on thedroplet microactuator as compared to droplet operations for the samedroplets including one or more specific reagents or mixtures on acorresponding droplet microactuator without the surfactant. In anotherrelated example, surfactants are preferably selected and provided in anamount which makes one or more droplet operations possible or morereliable for one or more droplets including amphiphilic molecules on thedroplet microactuator as compared to droplet operations for the samedroplets including amphiphilic molecules on a corresponding dropletmicroactuator without the surfactant.

In a preferred embodiment, the surfactant is added to the filler fluidin an amount which ranges from about 0.001 to about 10% w/w, or about0.001 to about 1% w/w, or about 0.001 to about 0.1% w/w. For example, inone embodiment the filler fluid is 2 cSt silicone oil and the surfactantis Triton X-15 in an amount which ranges from about 0.001 to about 10%w/w, or about 0.001 to about 1% w/w, or about 0.001 to about 0.1% w/w.The solid-liquid interfacial tension may be adjusted to control thewetting of the filler fluid on the droplet microactuator surfaces, forexample, to control the formation, thickness or behavior of thin filmsof the filler fluid between the droplet and droplet microactuatorsurfaces or to control the wetting behavior of the fluid when filling oremptying it from the droplet microactuator.

By doping filler fluid with surfactant, it was discovered that it ispossible to increase the concentrations of compatible protein solutionsby more than 3 orders of magnitude, from mg/L to mg/mL. The inventorswere able to reliably dispense and transport 25 nL droplets of 75 mg/mLlysozyme solution using the new filler fluid. For example, the fillerfluid may be silicone oil doped with a surfactant, such as Triton X-15.Preferably the surfactant is a lipophilic surfactant. In one embodiment,0.1% (w/w) Triton X-15, a lipophilic surfactant, was added to the oil sothat high concentrations protein droplets could be formed or dispensedfrom on-chip reservoirs. Droplet transport for all compatible fluids isfast (typically about 3-10 cm/sec) and reliable (>25,000 operations). Inone embodiment, the filler fluid includes a surfactant dopant in anamount which results in an increase in the concentration of a proteinthat can be reliably dispensed on the droplet microactuator.

The filler fluid may be selected to have a particular viscosity orvolatility. For example, a low viscosity liquid (e.g. 0.65 cSt. Siliconeoil) facilitates transport of droplets while a low volatility fillerfluid (e.g., 2, 5 or 10 cSt. Silicone oil) may be desirable to preventloss of filler fluid by evaporation. In some applications, evaporationof the filler fluid can be desired, so a low volatility filter fluid maybe selected. The filler fluid may be selected to have a particularviscosity dependence on temperature, since the viscosity of the fillerfluid affects the fluid dynamics and the temperature on the dropletmicroactuator may vary.

The filler fluid may be selected to have particular electricalproperties. For example, certain applications including electrowettingfavor the use of a filler fluid that is non-conductive (e.g., siliconeoil). Or the dielectric permittivity can be selected to control thecoupling of electrical energy into the system from external electrodes.In certain applications a non-conductive filler fluid can be employed asan electrical insulator or dielectric in which the droplet floats justabove the electrodes without physically contacting them. For example, inan electrowetting system a layer of filler fluid (e.g., silicone oil)between the droplet and electrode can be used to provide electrostaticcontrol of the droplet. Filler fluids may be deionized to reduceconductivity.

The filler fluid may be selected to have a particular density relativeto the droplet phase. A difference in density between the two phases canbe used to control or exploit buoyancy forces acting upon the droplets.Examples of two-phase systems useful in this aspect of the inventioninclude water/silicone oil, water/flourinert, and water/fluorosiliconeoil. When one phase is buoyant, then that effect can be exploited in avertical configuration as a means to transport one phase through theother. For example, a waste or collection well can exist at the top orbottom of the droplet microactuator where droplets are delivered to thatreservoir by simply releasing them at an appropriate point and allowingthem to float or sink to the target destination. Such an approach may besuitable for use in removing reactant from a droplet microactuator, e.g.removing fluid containing amplified nucleic acid for use in otherprocesses. Density differences can also be used as a means to control orengineer contact between the droplets and droplet microactuatorsurfaces. For example, a droplet not normally contacting a top-plate canbe released to sink or float to that surface to contact it. Densitydifferences and buoyancy effects can also be exploited for sensingapplications in which the movement of droplets is detected and relatedto a change in position, orientation or acceleration.

The filler fluid is selected for material compatibility with the dropletmicroactuator surfaces. For example, certain filler fluids can etch,dissolve, contaminate, absorb into or otherwise be incompatible withcertain droplet microactuator materials. For example, fluorinatedhydrocarbons, such as Fluorinert, may be incompatible with Teflon AF orCytop surfaces because of their tendency to dissolve these materials,while silicone oils may be incompatible with PDMS surfaces due to thetendency of these materials to dissolve each other.

The invention may include means for controlling the introduction orcirculation of the filler fluid within the droplet microactuator,cartridge and/or system. In one mode of operation the filler fluid isinjected once during the initialization of droplet microactuatoroperation. The filler fluid may be provided from an external sourceusing a syringe, dropper, pipettor, capillary, tube or other means.Alternatively, the filler fluid may be provided from a reservoirinternal to the droplet microactuator assembly or cartridge. As anexample, the fluid can be contained within a sealed pouch which ispunctured or compressed to transfer the liquid into the dropletmicroactuator.

In another mode of operation a means can be provided for multipleintroductions or recirculation of one or more filler fluids within thedroplet microactuator. A secondary fluid-handling system can be providedto inject and to remove fluid from within the droplet microactuator.Pressure, gravity or other means such as the use of thermal gradientscan be used to transport the filler fluid into or out of the dropletmicroactuator. Such a system can, for example, be used for the followingpurposes:

-   -   (1) To replenish filler fluid lost to evaporation or leakage        over time. A slow steady flow or periodic injection of filler        fluid can be employed to make up for any loss of filler fluid        volume.    -   (2) To provide “clean” filler fluid either continually or        periodically to reduce contamination between droplets. The        filler fluid can be cleaned either by completely replacing it or        by circulating it through a filter or bed of absorbent material        selected to remove contaminants.    -   (3) To provide a means for transporting droplets to waste. For        example, at the end of an assay, droplets can be released and        allowed to flow with the filler fluid to the outlet providing a        means to “flush” the droplet microactuator. Flushing the droplet        microactuator can be performed to reset the status of the        droplet microactuator in preparation to perform additional        assays.    -   (4) To exchange the filler fluid when different fluids may be        desired for certain steps, for example to replace oil with air        to allow drying of droplets, or to replace one oil with a        different oil.    -   (5) To provide a means of controlling the temperature of the        droplets by heating or cooling the fluid as it is circulated        through the droplet microactuator. The temperature of the filler        fluid entering and leaving the droplet microactuator can be        directly measured and the temperature and flow rate of the        filler fluid can be adjusted to provide optimal temperature        control inside the droplet microactuator.

Local regions of filler fluid or even individual units of filler fluidfor each droplet can be used. For example aqueous droplets can beencapsulated in an individual shell of fluid, such as oil, which movesalong with that droplet. Each such droplet would then have its own localfluid bath with the space between encapsulated droplets filled withthird immiscible liquid such as air or fluorosilicone oil. This approachcan be used to minimize the transfer of material between droplets in thesystem by partitioning into the oil phase while retaining theadvantageous properties of the oil with respect to evaporation andfouling of the surface. The shells of oil can be created by simplymoving the droplet through an oil interface, pinching off a unit of oilas the droplet creates a bulge along the interface.

Hybrid systems can be implemented in which different regions of thedroplet microactuator are filled with different fluids. For example,samples can be processed under oil and then transported into an airportion to be evaporated for subsequent analysis by MS. Conversely, asample can be collected in air and then processed under oil.

Magnetically responsive beads can be used to move material between oiland water phases on a droplet microactuator. Generally, water-solublecompounds or materials tend to remain within the droplets, unable tocross the oil-water meniscus in significant quantities, and oil-solublecompounds or materials remain in the lipophilic filler fluid. When thematerial is attached to magnetically responsive beads, a magnetic fieldmay be used to move the beads and attached material across the oil-waterboundary. The beads need to be selected such that they have sufficientaffinity for oil and water so that they can readily cross the meniscus.This operation is useful for drying or concentrating materials and canalso be used to facilitate washing and/or dilution. For example,material bound to a magnetically responsive bead can be removed from onedroplet and transferred by way of the filler fluid to another droplet.

Filler fluid can be circulated through the droplet microactuator toreduce contamination during and/or between runs. Filler fluid can becontinually or periodically flowed through the droplet microactuator, sothat fresh filler fluid is constantly supplied to the dropletmicroactuator. In addition to removing contaminates contaminated oil,this technique could be used at the end of a run to clear droplets fromthe array by removing the voltage so that droplets are released and flowwith the oil to an exterior of the droplet microactuator and/or into awaste reservoir.

8.8.5 Droplet Microactuator Loading

The droplet microactuator as contemplated herein generally includes oneor more input ports for the introduction of one or more filler fluids,reagents and/or samples (e.g., reagents and/or samples for conductingprotocols and/or assays as described elsewhere herein) into the dropletmicroactuator. In some embodiments, samples or reagents are loaded viathe input ports using conventional robotics. In one alternativeembodiment, droplets of sample or reagent are separated by plugs of oilin a long pre-loaded capillary (e.g., a glass capillary) which whenconnected to the droplet microactuator allows droplets of sample orreagent to be captured and routed on the droplet microactuator as theyare pumped out of the capillary into the input port. Another loadingtechnique involves pre-stamping reagents onto the droplet microactuatorand allowing them to dry, e.g., using a high-speed reagent stamping orprinting process. Yet another approach involves the use of a directplate-to-droplet microactuator interface in which the contents ofplates, e.g., 1536 or 384 or 96 well plates, are transported onto thedroplet microactuator in parallel by using pressure to force thecontents through input ports aligned with wells. Loading hardware may insome embodiments be electronically coupled to and controlled by thecontroller.

The droplet microactuator can be associated with or coupled with afluidic input module for loading and storage of sample and/or reagent.For example, a basic input module allows samples to be loaded using apipettor or other device. The system may be programmed to subdivide anddispense input fluid as discrete droplets which can be transported onthe control electrodes networks or pathways.

8.8.6 Reservoirs

The droplet microactuator as contemplated herein may include variousreservoirs (sometimes referred to herein as “wells”), such as inputreservoirs and/or processing reservoirs.

8.8.6.1 Input Reservoirs

In some embodiments, the droplet microactuator includes one or moreinput reservoirs (also referred to as “loading wells”) in fluidcommunication with one or more input ports, typically in direct fluidcommunication with the input ports. The input reservoir(s) serve asreservoirs for storage of bulk source material (e.g. reagents orsamples) for dispensing droplets (e.g. reagent droplets or sampledroplets). Thus, the input reservoir(s) may, for example, serve assample wells or reagent wells.

The input reservoirs generally include one or more well reservoirsdefining an interior space and an opening. The interior space defined bythe well walls is at least partially isolated by the well walls from theremainder of the interior of the droplet microactuator. The reservoirmay be adjacent (in any direction, e.g., vertically or laterally) to aport suitable for introduction of fluid from an exterior of the dropletmicroactuator into the input reservoir. One or more openings in thereservoir walls may be provided to enable fluid communication with theinterior volume of the droplet microactuator for dispensing of dropletsinto this interior volume. The opening(s) may permit fluid to flow or betransported into the interior volume of the droplet microactuator ontothe path or network of electrodes. Input reservoirs may also include oneor more vents for permitting displacement of filler fluid from the inputreservoir as fluid is introduced into or removed from the well via theport or the opening.

The input reservoirs may further include one or more planar controlelectrodes in a top or bottom plate adjacent to or within the spacedefined by the well walls. The planar electrodes can be electronicallycoupled to and controlled by the controller. In a preferred embodiment,the planar electrode has two or more branches or rays, such thatactivation of the control electrode during droplet dispensing in thepresence of a fluid exerts a “pull” on the fluid in a direction which isgenerally opposite to the direction of droplet dispensing. In somecases, the shape of the electrode results in a multi-vector pull havinga mean vector which has a direction generally opposite to the directionof the droplet being dispensed.

Well walls may, for example, be formed by protrusions from the top orbottom plates, and/or may be formed by deposition of a wall-formingmaterial on a surface of the top or bottom plate. For example, wellwalls may be formed from a soldermask material or polymeric gasketmaterial deposited and patterned on the surface. In some embodiments asource of continuous or semi-continuous sample or reagent flow iscoupled in fluid communication with one or more of the input ports.

It should be noted that while droplet dispensing may be conducted fromdefined reservoirs, in some embodiments, droplet dispensing is conductedwithout the use of physically defined reservoirs. Dispensing may proceedfrom source droplet which is confined during droplet dispensing, e.g.,by electrowetting forces or by hydrophilic surfaces.

8.8.6.2 Processing Reservoirs

The droplet microactuator may also include one or more processing wells,areas, or reservoirs. These reservoirs serve as a location for executingvarious droplet processing steps, such as mixing, heating, incubating,cooling, diluting, titrating, and the like. The droplet microactuatorincludes one or more paths or networks of control electrodes sufficientto transport droplets from the one or more input ports to the one ormore processing reservoirs. In some cases the processing reservoirs aresimply components or sections of these paths or networks. In otherembodiments, the processing reservoirs are defined processingreservoirs. Such reservoirs may, for example, be structured generally inthe same manner as the input reservoirs described above. However, theprocessing reservoirs are typically not in direct fluid communicationwith the input ports, i.e., droplet transport along the one or morepaths or networks of control electrodes is required add reagent orsample to the processing reservoir(s). In some cases, the processingreservoirs include a path or network of reservoirs therein to permitdroplet operations within the processing reservoirs. Otherconfigurations of the reservoirs or wells of various aspects of thepresent invention are discussed hereinabove with reference to Section8.4.1.

Mixing or dilution ratios can be established programmably by controllingthe number and distribution of constituent droplets delivered to eachreservoir. Furthermore, liquid which has been mixed within a reservoirmay be subsequently dispensed from that reservoir in the form ofunit-sized droplets for transport to another reservoir, for example, toperform serial dilution assays.

8.8.7 Thermal Control

The droplet microactuator of the invention may include a means forcontrolling the temperature of the droplet microactuator or a region ofthe droplet microactuator. Among other things, thermal control is usefulfor various protocols requiring heating or cooling steps. Examplesinclude amplification protocols requiring thermal cycling and variousassays that require incubation steps.

8.8.7.1 Thermal Control Designs

In general, thermal control may be provided in three ways: (1) thermalcontrol of the entire droplet microactuator; (2) thermal control of aregion of a droplet microactuator using a heater that is in contact withor in proximity to the controlled region; and (3) thermal control of aregion of the droplet microactuator using a heater that is integratedinto the droplet microactuator (e.g., in the substrate comprising thepath or array of electrodes and/or in a top plate of the dropletmicroactuator, when present). Combinations of the foregoing approachesare also possible.

In an integrated heater approach, temperature zones can be created andcontrolled using thermal control systems directly integrated into thedroplet microactuator. Integration of thermal control through thin-filmheating elements fabricated directly on the droplet microactuator isalso useful to maximize the speed, throughput and quality ofamplification reactions on the droplet microactuator. Due to their smallthermal mass, droplets can be thermally cycled extremely rapidly.Thermal control is enhanced by locating the heating elements proximateto the droplets and reducing the parasitic thermal losses between theheater and the droplet. Heating elements can be integrated into the topplate and/or bottom plate of the droplet microactuator.

Integrating heating elements onto the droplet microactuator also enablesthe use of multiple distinct thermal zones within the dropletmicroactuator. This permits multiple steps in an analysis, such assample preparation and thermal cycling, requiring different temperaturesto be performed simultaneously on different portions of the dropletmicroactuator. Droplets can be physically transported or “shuttled”between zones of different fixed temperatures to perform the thermalcycling aspects of the amplification reaction. This approach can produceeven faster reactions, since heating and cooling of the entire thermalzones is no longer rate-limiting. Instead, heating and cooling rates aredetermined by the time required to transport the droplets between thezones and the time required for the droplet temperature to equilibrateto the temperature of the zone once it arrives within the zone, both ofwhich are expected to be very fast. A further advantage is that reactionsteps can be “queued” rather than “batched” to permit greateroperational flexibility. For example, discrete samples can becontinuously fed into the droplet microactuator rather being deliveredat a single point in time.

Droplets may be thermally cycled in batch mode using a single heater orin flow-through mode by circulating the droplets through distincttemperatures zones created by the heating elements. The essentialdifference between batch and flow-through modes is that in batch modethermal control is effected by varying the temperature of the heaterwhile in flow-through mode, thermal cycling is effected by transportingthe droplets among distinct constant temperature zones. In the “batch”method a single integrated thin-film heater on the droplet microactuatorwas used to thermally cycle static droplets located within the heaterzone. In the “flow-through” method, two distinct fixed temperature zoneswere created on the droplet microactuator and thermal cycling wasperformed by shuttling the droplets between the two zones.

In the “batch” case, the thermal mass of the heater itself as well asthermal losses may be minimized through the use of thin-film heatersplaced directly adjacent to the droplets. Because the thermal masses,including the droplet itself, are so small, rapid temperature changescan be effected. Passive cooling (in filler fluid) is also rapid becausethe total energy input into the system is extremely small compared tothe total thermal mass.

For “flow-through” heating, a larger thermal mass is desirable becauseit helps to stabilize the temperature while a slower ramp rate istolerable because the heater temperature is not varied once it reachesits set point. A flow-through system can, for example, be implementedusing block heaters external to the droplet microactuator which weremore accurate and easier to control than thin-film heaters although, inprinciple either type of heater could be used to implement eithermethod.

In another embodiment, temperature is controlled by flowing orrecirculating heated filler fluid through the chip and around thedroplets.

The droplet microactuator layout is scalable, such that a dropletmicroactuator may include a few as one heating zone up to tens, hundredsor more heating zones.

8.8.7.2 Heater Types

Heaters may be formed using thin conductive films. Examples of suitablethin films include Pt heater wires and transparent indium-tin-oxide(ITO). ITO provides better visualization of the droplets for real-timeobservation. A remotely placed conventional thermocouple (TC) fortemperature regulation can also be used. In one embodiment, tiny metal(e.g., copper) vias in the PCB substrate are used to create tightthermal junctions between the liquid and the remote TC. Further, sampletemperature can be determined by monitoring the copper via using asurface mount thermistor or an infrared sensor. One advantage of using athermistor is that they are small enough (2×2 mm) to be soldereddirectly on the droplet microactuator, while an advantage of using IR isthat it is non-contact method which would simplify the interfacing.Because the thermal conductivity of copper is at least 700 times greaterthan the FR-4 substrate (350-390 W/m·K versus 0.3-0.5 W/m·K) thetemperature of a Cu via will accurately represent the temperature insidethe liquid. Heaters may be integrated on the bottom and/or top (whenpresent) plate of the droplet microactuator and on the bottom and/or topsurface of either plate, or integrated within the structure of eitherplate.

In one flow-through embodiment, reduced thermal gradients can beprovided by using heaters to create a continuous temperature gradientacross the droplet microactuator (e.g., from 100 to 50° C.). The use ofa continuous gradient will eliminate the need to overcome the steeptemperature gradients found along the edge of the heater blocks. Acontrolled temperature gradient would also significantly enhance thefunctionality of the device by allowing protocols with arbitrary numbersof temperature points to be implemented. Furthermore, each reaction canbe performed with a custom thermal protocol while only the temperaturesof the two or more blocks would need to be thermally regulated. Thedroplets will be transported to and held at the appropriate locationbetween the heaters to achieve a target temperature. The fluorescence ofthe droplets can be imaged using a fluorescence sensor as they aretransported over a detection spot. The temperature of the upper andlower target temperatures can be varied by changing the location of thedroplets.

In some embodiments, heaters located above the droplets may obscure thedroplets thus interfering with real-time optical measurements. In suchcases, the droplets can be transported out from underneath the heatersto a location which is preferred for optical detection (i.e. a detectionspot). Droplets may be periodically transported out from underneath theheaters to a detection spot on the droplet microactuator detectionpurposes, e.g. detection by fluorescence quantitation. Droplets may berouted into proximity with a sensor while cycling them from onetemperature zone to another.

8.8.8 Detection

The droplet microactuator systems as contemplated herein may includeon-chip and/or off-chip mechanisms for analyzing droplets. For example,the droplet microactuator may include one or more detection methods suchas amperometry, potentiometry, conductometry, absorbance,chemiluminescence, and fluorescence. The droplet manipulation module andthe detection module may in some embodiments be decoupled by buildingthem on separate substrates. Alternatively, the droplet microactuatormay incorporate detection components. Thus, for example, the dropletmicroactuator may include one or more of the following, on-chip oroff-chip: amperometry module arranged to measure current flowing througha droplet; potentiometry module including a measuring and a referenceelectrode arranged to measure equilibrium electrode potential of adroplet; conductometry module arranged to measure conductivity of adroplet; absorbance module arranged to measure energy or lightabsorbance of a droplet; chemiluminescence module designed to measurelight emission by chemical species in a droplet; fluorescence moduledesigned to excite and measure fluorescence of the species in a droplet.Off-chip detection modules may, for example, be provided in a cartridgethat comprises the chip and/or in an analyzer into which the cartridgeor chip may be inserted.

Preferred detection methods are absorbance, electrochemical,fluorescence, and chemiluminescence. In one embodiment, two or more ofthese methods are accomplished on a single droplet microactuator. Inanother embodiment, the droplet microactuator includes one detectionmodule, but the system is programmed to conduct more than one test usingthe module. In this embodiment, processed sample droplets requiringtesting are sequentially moved into position for testing. Thus, multiplesamples are multiplexed over a detection spot where a single detector isused.

Preferred assays for inclusion on the droplet microactuator includecalorimetric assays (e.g., for proteins), chemiluminescence assays(e.g., for enzymes), enzymatic and electrochemical assays (e.g., formetabolites, electrolytes, and gases), and conductometric assays (e.g.,for hematocrit on plasma and whole blood). In one embodiment, a singledroplet microactuator includes modules for 2, 3, 4, 5 or more differentkinds of assays. In another embodiment, a single droplet microactuatorcartridge includes on-chip and/or off-chip modules for 2, 3, 4, 5 ormore different kinds of assays. In yet another embodiment, a singledroplet microactuator system includes on-chip, off-chip, on-cartridgeand/or off-cartridge modules for 2, 3, 4, 5 or more different kinds ofassays.

8.8.9 Droplet Operations

The droplet microactuator may conduct various droplet operations withrespect to a droplet. Examples include: loading a droplet into thedroplet microactuator; dispensing one or more droplets from a sourcedroplet; splitting, separating or dividing a droplet into two or moredroplets; transporting a droplet from one location to another in anydirection; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletmicroactuator; other droplet operations described herein; and/or anycombination of the foregoing.

Droplet dispensing refers to the process of aliquoting a larger volumeof fluid into smaller droplets. Dispensing is usefully employed at thefluidic interface, the input reservoirs, and at processing reservoirs.Droplets may be formed by energizing electrodes adjacent to the fluidreservoir causing a “finger” of fluid to be extended from the reservoir.When the fluid front reaches the terminal electrode, the intermediateelectrodes are de-energized causing the fluid to retract into thereservoir while leaving a newly-formed droplet on the terminalelectrode. As previously noted, one or more electrodes in the reservoirmay also be energized to assist in separating the droplet beingdispensed from the bulk fluid. Because the droplet conforms to the shapeof the electrode, which is fixed, excellent accuracy and precision areobtained. Droplet dispensing is controlled by the controller. In someembodiments the invention employs droplet dispensing structures and/ortechniques described in U.S. Pat. No. 6,911,132, entitled “Apparatus forManipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat.Nos. 6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and 6,565,727, entitled“Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24,2000, both to Shenderov et al., the disclosures of which areincorporated herein by reference.

In some embodiments, droplet operations are mediated by electrowettingtechniques. In other embodiments, droplet operations are mediated byelectrophoresis techniques. In still other embodiments, dropletoperations are mediated by electrowetting techniques and byelectrophoresis techniques.

In one embodiment, separations may be performed using a combination ofelectrowetting and electrophoresis. Electrowetting microactuation can beused to create a channel to perform electrophoresis; to deliver a sampleto the channel or capture a sample fraction from channel following anelectrophoretic separation. For example, for forming a channel,electrowetting can be used to deform (stretch) a droplet of separationmedium in a long thin shape followed. In some cases, the channel may bepolymerized, e.g., using UV polymerization. In other cases, the channelmay be formed by using droplet operations to add droplets into aphysically confined microchannel. In a related embodiment, the effectivelength of an electrophoresis channel can be increased by capturing thefraction of interest in a droplet at the output and then returning it tothe input in a cyclical fashion. Using the same principle, a series ofprogressively finer separation can be performed. Separations may also beaccomplished using multiple different separation mediums at the sametime.

Droplet splitting or dividing of droplets generally involves separatinga droplet into two or more sub-droplets. In some cases, the resultingdroplets are relatively equal in size.

Transporting involves moving a droplet from one location to another inany direction. Droplets may be transported on a plane or in threedimensions. It will be appreciated that a variety of droplet operations,such as dispensing and/or splitting may include a transporting element,in which on droplet is transported away from another droplet.

Merging involves combining two or more droplets into a single droplet.In some cases, droplets of relatively equal size are merged into eachother. In other cases, a droplet may be merged into a larger droplet,e.g., combining droplet with a larger volume present in a reservoir.

Mixing a droplet involves various droplet manipulations, such astransporting or agitating, that result in a more homogenous distributionof components within the droplet. In one mixing embodiment, a dropletpositioned over an electrowetting electrode is rapidly and cyclicallydeformed in place by activating and deactivating the electrode, inducingfluid currents within the droplet which facilitate mixing.Frequency-dependent effects such as mechanical resonances may be used totune the quality and speed of mixing. Compared to techniques whichrequire transport of droplets on a surface for mixing this approachminimizes the area required for mixing. This mixing scheme can beemployed without the presence of a top plate. Due to space-savingadvantage, this scheme could provide for simplified mixing in reactionwells since only one electrode is needed.

Reagents or samples from reservoirs may be dispensed as discretedroplets for transport to other locations on the droplet microactuator.

The invention includes droplet operations using droplets comprisingbeads. A variety of such operations are described elsewhere herein. Inone embodiment, beads are used to conduct droplet operations on reagentsthat are prone to interfere with droplet operations. For example,certain proteins may be prone to bind to surfaces of a dropletmicroactuator and/or to partition into the filler fluid. Immobilizingsuch compounds on hydrophilic beads can be used to facilitate dropletoperations using the compounds. The compounds can be bound to the beads,and the beads can contained with a droplet which is subjected to dropletoperations.

In one particular dispensing operation, coagulation is used to separateserum from whole blood. Whole blood is loaded onto the chip and combinedwith a droplet comprising a coagulating agent. Following coagulation,droplets are dispensed from the sample. Because cells and platelets aretrapped in place, the liquid dispensed from the sample will contain onlyserum.

8.9 System

The various aspects of the present invention may also include systemswhich include the droplet microactuator coupled to a processor which maybe programmed to control the droplet microactuator. Various input means,such as keyboards, switches and touch screens, and various output means,such as display screens, output ports, and wireless transmittingdevices, may also be included in electronic communication with theprocessor. The droplet microactuator may be presented as a component ofa cartridge for insertion in an analyzer. The cartridge may include oneor more pre-loaded reagents, which are dispensed into the dropletmicroactuator prior to, during, or after insertion of the cartridge intothe analyzer.

Systems can be programmed to execute a wide variety of protocolsinvolving any number of droplet manipulations. Multiple droplets can beindependently and simultaneously manipulated on a single dropletmicroactuator. The capacity to independently manipulate multipledroplets in parallel enables execution of complex protocols as a seriesof basic microfluidic instructions. Moreover, droplet microactuators arescalable, enabling systems that control tens, hundreds, thousands ormore parallel droplet manipulations per droplet microactuator chip. Forexample, at any one moment, up to a maximum of every control electrodeon the droplet microactuator may be engaged in a droplet operation.

The system can be programmed to enable users to input instructions forthe execution of protocols. Existing protocols may be monitored andadjusted according to user requirements. Complex protocols can beimplemented in which the outcome of one or more steps determines theselection of one or more subsequent steps. For example, a droplet inwhich a certain measured result is positive may be transported forfurther processing, while a droplet in which a result is negative may bediscarded, or vice versa.

Flexibility of operations in the systems of the invention is muchgreater, for example, than the flexibility of robotic systems, whichwould require a massive assembly of robotics, a huge facility, andthousands of times the amount of reagents to achieve anything near themassively parallel operations that are enabled by the dropletmicroactuator. Nevertheless, in some embodiments, robotics may be usefulfor droplet microactuator or cartridge placement, reagent loading,placement of detectors for external measurements of on-chip phenomena,and the like.

8.10 Kit

A further aspect of the invention is a kit including reagents, samplecollection devices, and/or a droplet microactuator or cartridge forconducting the methods of the invention.

9 LITERATURE CITED

The entire disclosure of each of the following references isincorporated herein by reference:

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10 CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention.

This specification is divided into sections for the convenience of thereader only. Headings should not be construed as limiting of the scopeof the invention.

It will be understood that various details of the present invention maybe changed without departing from the scope of the present invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

1. A droplet actuator for providing an array of crystallizationconditions, the droplet actuator comprising: (a) two or more processingreservoirs; (b) two or more dispensing reservoirs collectivelycomprising two or more crystallization reagents; and (c) a port forintroducing a sample for crystallization analysis.
 2. The dropletactuator of claim 1 wherein the crystallization reagents comprise coarsegrid reagents.
 3. The droplet actuator of claim 1 wherein thecrystallization reagents comprise fine grid reagents.
 4. The dropletactuator of claim 1 wherein the crystallization reagents comprise one ormore reagents selected from the group consisting of salts, pH buffers,precipitants, and water.
 5. The droplet actuator of claim 1 wherein thesample comprises a protein or peptide.
 6. The droplet actuator of claim1 wherein the sample comprises a small molecule.
 7. A system comprisingthe droplet actuator of claim 1 programmed to conduct droplet operationssufficient to create an array of crystallization conditions for thesample on the droplet actuator.
 8. A method of providing an array ofcrystallization conditions, the method comprising: (a) providing adroplet actuator comprising: (i) two or more processing reservoirs; (ii)two or more dispensing reservoirs collectively comprising two or morecrystallization reagents; and (iii) a port for introducing a sample forcrystallization analysis; and (b) executing droplet operations on thedroplet actuator to combine crystallization reagents with sample in theprocessing reservoirs to create two or more crystallization conditionsin the processing reservoirs.
 9. A method of identifying crystallizationconditions, the method comprising: (a) providing an array ofcrystallization conditions according to the method of claim 8; (b)permitting the crystallization conditions to remain intact for apredetermined period of time; and (c) identifying wells in whichcrystallization results.